Strategies and Methods for Protein Detection

#### **Chapter 1**

## Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial Expression

*Kubra Acikalin Coskun, Nazlıcan Yurekli, Elif Cansu Abay, Merve Tutar, Mervenur Al and Yusuf Tutar*

#### **Abstract**

Protein purification is not a simple task. Yet, overexpression at bacterial systems with recombinant modifications brings further difficulties. Adding a tag, an affinity label, and expressing particular domains of the whole protein, especially hydrophobic sections, make purification a challenging process. Protein folding pattern may perturb N- or C-terminal tag and this terminal preference may lead to poor purification yield. Codon optimization, solvent content and type, ionic conditions, resin types, and self-cleavage of recombinant proteins bring further difficulties to protein expression and purification steps. The chapter overviews problems of protein purification through a small peptide overexpression in bacteria (Recombinant anti-SARS Coronavirus 2 (SARS-Cov-2) Spike protein Receptor Binding Domain (RBD) antibody (Clone Sb#14). The chapter also covers troubleshooting at distinct steps and highlights essential points to solve crucial issues of protein purification.

**Keywords:** protein, prokaryotic protein expression, and purification, protein modeling, protein aggregation, ionic strength

#### **1. Introduction**

Recombinant DNA technology involves genetic engineering; cutting DNA molecules from distinct biological species and then ligating them to a vector for expression [1, 2]. The technology helps to express the desired protein in large quantity rather than extracting from bulk amounts of tissues and animal fluids. Proteins are synthesized and modified depending on their functions in an organism. As an initial step, DNA encodes protein through transcription from mRNA synthesis. Then, mRNA is converted into protein. Transcription and translation occur simultaneously in prokaryotic organisms. The conversion of mRNA to protein begins before the synthesis of the mature mRNA transcript [3]. Protein expression involves the synthesis, modification, and regulation of a particular protein in a living organism. However, bacterial systems lack human protein modifications but overexpress recombinant proteins in bulk amounts [4]. Recombinant protein expression is useful

to understand the structure and function of proteins. A network of protein complex functions can be distinguished by the characterization of individual proteins function as well as interactions through recombinant protein techniques. Protein–protein and protein-ligand interactions may be highlighted by expressing interacting domains and by introducing key mutations to reveal key domains and residues, respectively. Considering the size and complexity of proteins, protein production is very efficient with vector templates using K12 bacterial systems [2, 4].

#### **1.1 Bacterial protein synthesis**

Recombinant protein production in bacterial systems is fast, easy, and highly efficient [5]. The general strategy for recombinant protein production involves transforming the cell with a DNA vector containing the open reading frame of the gene, then after subcloning to the expression system, the protein is induced in the cells. After incubating the induced cells, harvested cells are lysed for further separation. The selection of the purification system depends on the type of protein, the affinity tag of the plasmid, the isoelectric point (pI) of the protein of interest, the molecular mass of the protein, the targeted yield, and the degree of functional activity. The lysed cells are purified through column chromatography with a proper resin(s) and a convenient buffer system [6, 7]. But in practice, several steps may cause problems. These include inadequate growth of the selected bacterial host cell, the formation of inclusion bodies, protein aggregation, structural alteration, recombinant protein nonspecific interaction with cellular proteins, problems in colon systems used in purification [6, 8]. Further, as eukaryotic proteins expressed in E. coli may not perform post-translation modifications of other organism proteins, loss of function is observed. In addition, some of the proteins expressed are exposed to hard-denaturing agents or may cause collapses in structure and this often results in insolubility problems. Overexpression of recombinant proteins in bacterial systems leads to the formation of inclusion bodies. Re-folding of these proteins into their bioactive forms is cumbersome and requires a variety of agents and processes [1, 9].

#### *1.1.1 Host cell selection*

First of all, the choice of the host cells to produce intact protein in the synthesis mechanism forms the mainline of the whole system. Microorganisms used in recombinant protein expression systems include bacteria and yeast. Each host has strengths and weaknesses. The organism to be selected varies depending on the particular protein, working conditions, desired yield. For example, if the desired protein has post-translational modifications, choosing a prokaryotic expression system would not be proper [10]. BL21 (DE3) and its derivatives are by far the most commonly used strains for recombinant protein synthesis. In addition, its genetics are characterized in more detail than any other microorganisms. Recent studies suggest that BL21 (DE3) gene-level research made this bacterium more important for the production of heterologous proteins. This host cell provides maximum efficiency in protein expression through inexpensive substrates, capable of rapid and high-yield growth. A modified form includes a pLysS plasmid that encodes T7 lysozyme. This lowers the background protein expression of recombinant protein but does not perturb IPTG induction. The plasmid is especially useful in toxic cases and provides an option for protein over-expression. Yeast is an alternative recombinant protein production host and provides eukaryotic post-translational modifications with high yield.

Yeast growth temperature (30°C) is lower than that of bacteria (37°C) but the growth rate is much slower. Further, the transformation of plasmids to yeast is relatively difficult and the selection of transformed cells and growth conditions require special conditions [10, 11].

#### *1.1.2 Plasmid selection*

The expression plasmids consist of the replication origin, promoter, and multiple cloning sites. The most important issue to consider when choosing an appropriate vector is the copy number property. Because the number of copies is controlled by the replication. It is not always true to assume that the high amount of plasmid is proportional to the yield of recombinant protein expression. Because the high copy is inversely proportional to the rate of bacterial growth. In addition, this condition creates plasmid instability and creates a metabolic load. As a result protein production yield decreases [12, 13].

#### *1.1.3 Promoter*

Prokaryotes have to adapt to the environment by responding quickly to environmental changes. E. coli cells cannot use lactose directly as a source of carbon. But they use glucose, a component of lactose. For the bacterial cell to metabolize lactose, it is necessary to take lactose into the cell and break it down into a glucose monomer. For this, it is necessary to synthesize three different enzymes in the cell [6, 14]. As with E. coli, bacteria combine genes related to the same metabolic pathways to form clusters called operons. Transcription of the genes that make up the operon start from a single promoter. The resulting transcription product consists of an mRNA molecule containing information from multiple genes. Preserved DNA sequences in the promoter region help connect the enzyme to the DNA molecule. Induction is difficult in the presence of easily metabolized carbon sources. If lactose and glucose are present in the environment, expression from the lac promoter is not fully induced until all glucose is used up. In the absence of glucose, the promoter expresses the three enzymes to break down the lactose to obtain glucose. This property is used to induce prokaryotic expression vectors through IPTG (isopropyl 1-thio-β-d-galactopyranoside); a lactose analog that binds lac repressor [14, 15]. In the commercial vectors, IPTG starts the transcription of the lac operon and eventually induces protein expression where the gene of interest is controlled by the *lac* operator.

#### *1.1.4 Marker selection*

A resistance marker is added to the plasmid to prevent the growth of cells that do not carry plasmids. This can be achieved by using a selection marker. For example, ampicillin resistance is conferred by the *bla* gene, β-lactamase, a periplasmic enzyme that inactivates the β-lactam ring of β-lactam antibiotics [16].

#### *1.1.5 Affinity tags and its contribution to protein solubility*

The addition of affinity tags to the plasmid (such as His Tag, glutathione-Stransferase, and cellulose-binding domain) is employed to separate a particular protein from the heterogeneous protein mixture during purification, forming disulfide bonds, increasing the solubility of the recombinant proteins and transferring

them to the periplasm region. Affinity tags have a great role in separating the desired protein from cell lysate in recombinant protein purification. Affinity tags are divided into small peptide tags (amino acids) and large polypeptide tags (fusion partners) [17]. Small peptide tags are less likely to interfere when fused to the protein. In some cases, this may have negative consequences on the tertiary conformation and biological activity of the fused chimeric protein. Vectors are available that allow tags to be placed optionally at the N-terminal or C-terminal end. It is more advantageous to position a signal peptide at the N-terminal end for better secretion of the recombinant protein. At this point, it is important to know which end of the protein is embedded in the folding pattern by examining the three-dimensional structure of a particular protein, and it is necessary to place the label on the solvent-exposed end. Examples of small peptide tags are poly-His, c-Myc, and FLAG [18]. His-tagged proteins can be purified by affinity chromatography in resins containing positively charged metal ion nickel. In addition, at the end of purification, with commercial antibodies, labeled recombinant protein can be detected by western blot [17–21]. On the other hand, it increases the solubility of the recombinant protein produced by the addition of a nonpeptide fusion partner (large polypeptide label). The most commonly used fusion labels include Thioredoxin (Trx), Ubiquitin, SUMO, Maltose binding protein (MBP), Glutathione S-transferase (GST) [17, 22]. The reason why fusion partners show properties that increase the solubility of the protein is still not fully explained. Though, MBP label has been shown to carry a small chaperone activity. The GST label has been shown to have the weakest solubility-enhancing effect among fusion partners. Trx has the most solubility-enhancing properties, but due to its size, it may cause adverse effects. In recent years, studies have shown that "Calcium-Binding Protein Fh8" tag derived from a parasite called "Fasciola hepatica" recombinantly added to proteins increases protein solubility [6, 17, 20, 21]. Studies are underway for better solubility enhancing effect of recombinant protein tags.

#### **1.2 Troubleshooting strategies for recombinant protein expression**

Even if the effective parameters are provided in the production of recombinant protein, it may not be determined exactly whether the desired protein will be eluted excessively and in active soluble form. Therefore, there are additional strategies for optimizing protein expression [7].

#### *1.2.1 Low or no protein production*

If the desired protein cannot be detected using sensitive techniques or is detected at a low expression rate, the problem is usually caused by a toxic effect of the heterologous protein in the cell. As a result of protein toxicity in the host cell, cells cannot proliferate at a sufficient level and show a low growth rate [7, 23]. The first measure to solve this problem should be followed before proliferating cells are induced. If the growth rate of the recombinant cell is slower than that of the strain with empty vectors, it is related to either gene toxicity or the basal expression of toxic mRNA and protein. Control of basal production is associated with the operon system. *Lac*I or *LacI*Q expression blocks transcription in *Lac*-based promoters. High-copy plasmids must be cloned in the *Lac*I Q expression vector. Since the presence of tryptone or peptone in the growth medium contains inducing lactose, a more controlled expression is provided with the addition of glucose at 0.2–1 w/v. Plasmids containing T7-based promoters prevent leaky production, such as BL21DE3-pLYS (S) [8, 24].

*Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

#### *1.2.2 Limiting factors in the medium*

Luria Bertani (LB), the most commonly used growth medium environment for E. coli culture, is an ideal environment for high-nutrient cell growth. When recombinant protein production cannot be replicated with the recommended mechanisms, production efficiency can be increased by increasing the volume of the targeted protein. A successful result can be achieved with adequate ventilation with rigorous shaking of the growth medium. Although LB has a high protein content, cell proliferation is partially reduced. This is due to the low carbohydrate content of LB. As a solution to this situation, increasing peptone and yeast extract provides higher cell proliferation with the addition of MgSO4, which contributes to the sonic intensity of the environment. In addition, the amount of acid released as a result of increased glucose metabolism over time exceeds the buffering capacity of LB. In case of acidification of the growth medium, 50 mM phosphate salts can be added to the environment and buffered [7, 11]. In the broth culture, as the number of cells per unit media increases, oxygen limitation occurs and changes the metabolic capacity of the cell. This prevents optimal growth and the easiest way to increase the amount of oxygen in the growth medium is to increase the speed of the shaking containers. The optimum shaking speed range is 300–400 rpm. Several anti-foaming agents can be added to the broth culture to prevent the negative effect of the foams formed by strong shaking on oxygen circulation [24].

#### *1.2.3 Formation of inclusion bodies*

The inclusion bodies formed in E. coli are denatured protein molecules that do not display biological activity. Dissolving, refolding, and purification protocols should be applied, respectively, to make inclusion objects functionally active and soluble. In the transfer of a foreign gene to E. coli, control of gene expression is lost. The nascent polypeptide expression depends on several factors such as osmosis, folding pattern, and pH. If expression increases, the number of unspecific hydrophobic interactions in the polypeptide chain increases. This causes instability and clustering in poly peptization. The resulting protein aggregation is called "inclusion bodies." The main reason for the formation of clustering is due to the deterioration of the balance between protein aggregation and protein resolution [1, 25]. Therefore, a soluble recombinant protein can be obtained through strategies that eliminate the factors causing the formation of inclusion bodies. As mentioned in the "Affinity tags" section, one way to prevent the solubility problem that may occur in the expressed protein is; combining the desired protein with a fusion partner (large polypeptide tag) that acts as a solubilizer [17].

#### *1.2.4 Disulfide bond formation*

To obtain the biologically active three-dimensional structure of recombinant proteins, it is important to establish the right disulfide bonds. The formation of improper disulfide bonds causes the protein to fold incorrectly and the formation of inclusion bodies. Disulfide change reactions catalyzed by many enzymes in the Dsb family, where cysteine oxidation occurs in E. coli periplasm, form disulfide bonds in the polypeptide chain [26]. In the cytoplasm, the formation of disulfide bonds is rare because the remnants of cysteine are catalytic regions for many enzymes in the cytoplasm. The wrong disulfide bonds in these regions can cause protein inactivation, clustering, and incorrect folding. However, some strains of E. coli have conditions that trigger the formation of a disulfide bond [5].

#### *1.2.5 Addition of chemical chaperones and co-factors*

Molecular chaperones form the heart of protein synthesis and help nascent polypeptides fold into their active structures. Some specific types of chaperones, such as ClpB, can cleave unfolded polypeptides contained in inclusion bodies. However, high levels of recombinant protein production may result in increased molecular traffic in the cytoplasm, resulting in uncontrolled protein folding control. One strategy used to solve this problem is to arrest protein expression by removing the inducer after a centrifugation step and adding a fresh medium containing chloramphenicol, the protein expression inhibitor. Thus, it allows the recruitment of molecular chaperones to enable the folding of newly synthesized recombinant polypeptides [27, 28]. One of the systems used commercially for protein folding is chaperone plasmids. This system consists of plasmids that allow overexpression of different chaperones or their combinations. Examples of these are GroES-GroEL, DNAK/DNAJ/GrpE [27]. When proteins are released from inclusion bodies, denatured with urea, and subsequently folded *in vitro*, the addition of osmolytes (proline, trehalose) at a concentration ratio of 0.1–1M increases the yield of soluble protein. In addition, the correctly folded protein may require special cofactors such as metal ions (such as magnesium, iron/ sulfur) or polypeptide cofactors in the media medium to reach its final conformation. The addition of these compounds to the culture increases the yield and the folding rate of soluble proteins [8, 28].

#### *1.2.6 Slowing down the production rate*

Slowing the production rate of the recombinant protein reduces cellular protein concentration and protein trafficking, allowing the synthesized polypeptides to fold more smoothly. The most common method of reducing the rate of protein synthesis is to lower the incubation temperature [29]. Decreased temperature prevents the formation of aggregation due to its reduction of hydrophobic interactions. Recombinant protein synthesis occurs in the temperature range of 15–25°C. However, when working at the lower temperature range, this causes slower growth and therefore lower protein synthesis. This obstacle can be overcome with commercial products. The ArticExpress™ (Agilent Technologies) competent cells improve recombinant protein expression at low temperatures through co-expressing ortholog genes of E. coli GroEL and GroES from Oleispira Antarctica, namely Cpn60 and co-chaperone Cpn10. These chaperones work together to fold a substrate protein, and usually carry re-folding activity at 4–12°C temperature range, increasing recombinant protein yield and solubility at lower temperatures [30].

#### **2. Techniques used in recombinant protein purification and detection**

Selection of the purification methods generally uses distinct characteristics of the proteins. The distinct properties of recombinant proteins may include chemical, biological, and physical features due to differences in spatial structure and amino acid sequences. Usually, to benefit from these differences, multiple steps are required in the optimal purification process but it should be noted that each step may cause loss of product stability and/or yield, therefore the lowest number of steps are recommended overall for maximum yield. So, method selection determines the ratio of better yield to the better-purified product. Key factors that can affect the purification

#### *Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

selection steps include the solubility of the lysate, sample size, and physicochemical properties of the target protein. The first step for purification is to analyze the protein characteristics and match them with literature reports-protocols. For example, a useful parameter in the purification process is amino acid composition. pKa and pI values can be calculated using the amino acid composition. Determination of the values helps to select column type, buffer, pH, or resin type. Once optimization of the purification is established, the method may be employed for a protein with similar sequences or motifs at least for orthologs or isoforms [31]. The characteristic features of proteins that are mainly used for purification type selection are solubility, size, charge, and specific binding affinity [32]. By using these properties, numerous techniques may be employed in protein purification. Solubility parameter can be used with "salting out" through the knowledge of proteins mostly being less soluble in high salt concentrations. And hence, this strategy can be used to separate the protein of interest. Further, dialysis can be used after salting out to remove the salt molecules [33]. Another technique that uses size difference is gel-filtration or size exclusion chromatography (SEC). A column with porous beads resin is used for this and as the sample goes through the column, beads help to separate molecules. The beads are usually 0.1 mm in diameter, so bigger molecules cannot permeate the pores but small molecules penetrate into the pores and are trapped there for a while until the molecules exit again and return to solvent. This action retards small molecules but bigger molecules travel rapidly through a void volume with buffer flow. Small molecules shielding and bigger molecules faster flow separate molecules from each other in fractions depending on their sizes. And as the molecules exit the column, bigger molecules elute first and then, smaller molecules come after.

If the net charge is criteria to be used as a separating feature, ion-exchange chromatography can be used. If the target protein is positively charged as a cationic protein, then, a negatively charged carboxymethyl-cellulose (CM-cellulose) prepacked column/resin can be used. But if the protein is negatively charged as in anionic proteins, then positively charged diethylaminoethyl-cellulose (DEAE-cellulose) prepacked columns/resin can be used [33]. It is also known that proteins can have high affinities for certain chemical groups. Affinity chromatography can use this feature to purify proteins and its effect is the best on proteins with affinities to highly specific molecules.

Distinct separation techniques, that is, ion exchange and gel filtration may be employed at high-pressure liquid chromatography (HPLC) with proper column selection. This technique differs from the others because the applied pressure is significantly higher and it does not rely on gravity for sample flow. However, high-pressure limits the purification of higher molecular weight proteins as the pressure denatures protein structure. For higher-molecular-weight proteins FPLC (fast-pressure liquid chromatography) is preferred to prevent pressure-dependent denaturation. FPLC is the preferred technique for protein chemists since any target protein can be separated from cellular lysate readily. And the technique provides a wide range of column options. The flexibility of this technique provides the purification of stable proteins with a high yield. Lower pressure provides advantages as well. Clogging due to lysate content and backpressure problems are less likely encountered compared to that of HPLC. The techniques may also be used with tagged proteins. Histidine tag is one of the most common ones that are used with recombinant proteins and it has a high affinity of metal ions like Ni2+ [17]. To screen if the purification steps are working, gel electrophoresis can be used. In-gel electrophoresis, proteins are separated by their mass as they go through the gel and the smaller ones move faster. As they get

separated by their masses, proteins can be visualized in the gel and the gel show protein of interest among others. Another feature to separate proteins is their isoelectric point. This point represents the pH level where the protein has zero net charges. The technique that uses this property to separate proteins is called isoelectric focusing. When proteins go through a pH gradient gel, they will stop at the point where they have no net charge and get separated from the proteins with distinct isoelectric points. To get more specific results, isoelectric focusing can be coupled with SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in a technique called two-dimensional electrophoresis. In this technique, first isoelectric focusing is done as the sample goes through the gel horizontally and after proteins stop at their respective pH levels, vertical electrophoresis starts. So, the sample is separated according to the isoelectric points horizontally and by their masses vertically. The two-dimensional separation technique is employed to distinguish differences in two different states. Actually, distinct spots may be characterized by MALDI-TOF mass spectrometry.

#### **2.1 Protein structure determination and screening databases**

The structure and function of a protein are essential to characterize the linkage associated between common motifs and biochemical activity [34]. These features are normally determined by NMR or X-ray crystallography techniques [35]. NMR determines dynamic structure but the technique is limited to protein molecular mass. However, an instant picture of the protein structure with a relatively higher mass can be taken by X-ray crystallography. After elucidation of information from these structure determination techniques, scientists concluded that similar sequences show similar structural patterns [36]. Then, different databases which can display protein 2 and 3-dimensional structures have been developed. Determination of protein structure is important for not only understanding the function but also important for protein experiments, such as protein purification. Proteins have alpha helixes, turns, and beta sheets as secondary structures. Beta sheet structures and the outer surface of alpha helixes of proteins can accumulate within the cellular medium or stick to each other and other proteins during aggregation. *In vitro* experiments of proteins showed that proteins do not act like they are within the cell in terms of their stability, charge, and interactions properties. To understand the optimum conditions for protein purification, structure determination databases and pI calculation play crucial roles. In the case of His-tagged proteins, pI is one of the most important parameters for purification. pI is determined with a special calculation by considering amino acid sequences. NCBI Protein Data Bank provides the amino acid sequence of desired proteins (https://www.rcsb.org/). Several properties of the selected protein can be calculated by Expasy (Expert Protein Analysis System) Tool which is one of the most convenient tools on the web (https://web.expasy.org/compute\_pi/) [36].

#### **2.2 Swiss modeling and I-Tasser**

Protein structure can be determined by bioinformatic tools such as Swiss Modeling or I-Tasser. Swiss model and I-Tasser are Protein Data Bank (PDB) dependent protein homology modeling databases. They use the known templates from PDB. Swiss Modeling is quicker than I-Tasser, however, I-Tasser produces better and more stable results. Swiss Modeling uses known sequences on the internet and generates data by comparing known structures. Both Swiss Modeling and I-Tasser have the advantage

#### *Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

of understanding the main structure of the protein. Protein structure screening is a key factor to understanding interactions of proteins within themselves and with the environment. 3D modeled protein structures can be screened with commercially available tools like YASARA and Discovery Studio Tool. In these tools, not only protein structure is screened, but also domains of proteins can be separated, deleted and water molecules can be removed. UCSF Chimera and Autodock Tools also can be used for screening. After modeling, structures may be downloaded as PDB format and can be visualized by several programs: Discovery Studio, Autodock Vina, UCSF Chimera, etc. Interactions within the protein and secondary-tertiary structures can be obtained from these tools. All these tools work with a PDB file. There are other tools for protein structure determination and can be found at https://www.click2drug.org/. This website provides database links for distinct applications.

#### **3. Importance of databases and protein structure determination in recombinant protein purification**

Protein databases play a crucial role in bioinformatics and help to find information related to their research. In this way, all biological information becomes accessible through data mining tools saving time and resources. The first step in the study of a new protein is searching databases. Without the prior knowledge from such searches, previously known protein information could be missed, or an experiment could be repeated unnecessarily. There are hundreds of useful databases that can be used in protein research. However, in this study, the pI and 3D structure of the peptide were obtained using the Swiss database and Expasy Database. The purpose of the Swiss Database is to make protein structure modeling accessible. Therefore, with this database, the 3D shape of the protein provided us with information to understand the structure of the peptide and its interactions. Additionally, Expasy Database provides information about proteomics, post-translational modification prediction, primary, secondary, and tertiary

#### **Figure 1.**

*Predicted structure of Sb#14. Sb#14 is a recombinant synthetic monoclonal antibody 14 used to detect spike protein of COVID-19 and used for immunodetection of the virus. SB#14 is modeled by the Swiss model to design purification steps for the TUSEB project.*

structure analysis, sequence alignment, and pI of the protein [37]. The pI of the peptide provides the pH range where that peptide has a negative charge and prepares the buffer solution accordingly. Consequently, databases have key roles in biological research, and enormous data for protein structures, functions, and sequences can be generated by these available databases. These data offer essential information about our protein research as well. **Figure 1** provides the predicted structure for our research. SB#14 model indicates that the protein is formed from β-sheet structures.

#### **4. Structure- and design-based difficulties in recombinant protein purification**

#### **4.1 Protein insolubility**

Protein solubility is one of the most important protein properties and it can be defined as the protein concentration in a saturated solution that is in equilibrium with a solid phase [38]. Not only some extrinsic factors, including pH, ionic strength, temperature, and some solvent additives, can affect the protein solubility but also several intrinsic factors influence protein solubility. Moreover, the amino acids on the protein surface are the primary intrinsic factors that impact protein solubility [39]. Several studies have revealed the relationship between protein solubility and sequence-derived characteristics. Wilkinson and Harrison et al. provided a simple approach for predicting protein solubility from the sequence, which was further refined by Davis et al. [40]. The average charge, which is derived by the relative quantities of Asp, Glu, Lys, and Arg residues, and the concentration of turn-forming residues are the two parameters used in their solubility model (Asn, Gly, Pro, and Ser). In addition, Christendat et al. have demonstrated that insoluble proteins had more hydrophobic stretches (more than 20 amino acids), less glutamine (Q 4%), fewer negatively charged residues (DE 17%), and a higher percentage of aromatic amino acids (FYW >7.5%) than soluble proteins [41]. The affinity tag (His/ GST) in recombinant protein purified by affinity chromatography allows the protein to be purified. However, affinity tag has been observed to alter the biological activity of the protein. Because a minor difference affects protein solubility, the choice of affinity tag at the N- or C-terminus is important when expressing a protein domain. Klock and colleagues investigated a nested collection of 2143N- and C-terminal truncations from 96 targets and found significant variance in both solubility and aggregation processes by changing just a few amino acids in a protein length [42]. Therefore, it is essential to analyze which end of the protein is hidden. Furthermore, if the three-dimensional structure is known, the tag should be kept in a solvent-accessible end. In this way, the solubility of the protein can be increased. Insoluble proteins can aggregate during the expression process. That is why the different parameters should be optimized. On the other hand, during downstream purification steps, protein aggregation can occur. In these cases, developing a suitable and optimized purification procedure for each protein is critical. Sb#14 hydrophobic nature (**Figure 1**) leads to solubility problems as well as aggregation. The protein sticks to larger proteins and this led to difficulties in purification.

#### *4.1.1 Effects of imidazole on protein solubility*

Imidazole is one of the most widely used organic compounds in protein affinity purification processes. It is used as a competitive agent to elute the histidine-tagged proteins.

#### *Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

High concentrated imidazole that includes protein samples should be eliminated after eluting from the nickel column by dialysis [43]. In spite of all precautions, his tagged Sb#14 sticks to other proteins and has low solubility, therefore, SEC is used for protein purification rather than affinity purification. As mentioned, SEC separates proteins according to the molecular weight of the molecule. SEC performed with Superdex 75 size-exclusion column 10/30 (GE Healthcare, Princeton, NJ, USA). Moreover, SEC (15 cm length with r, 3 cm column) used in this study separates aggregates readily. Lower molecular weight of Sb#14 provides an advantage in the purification process, recalling that larger proteins elute first. This custom SEC column was unique as the resin resolution is high while the column length is relatively lower. The choice of resin and column size helped to resolve Sb#14 from bacterial lysate in a single purification step. The peptide (MW: 12.468 g/mol, pI: 8.91) is small and prone to aggregate. Therefore, the single-step purification blocks the self-cleavage of protein domains.

#### *4.1.2 Protein folding*

The stability of the protein in various buffer compositions and pH levels with and without ligands should be determined. There are some useful websites for fold recognition that can be used to predict the protein fold (PSI-BLAST and SEARCH). Some proteins are misfolded and require the addition of a cofactor, or ligand to restore proper folding and increase stability. For instance, beta-sheets are more prone to form amyloidlike aggregates if there are other binding partners that support protein stabilization and folding [44]. If the protein has a large number of beta-sheets, aggregation may be observed. This can be explained by the tendency of sticking together at Sb#14 and leading to the formation of insoluble aggregates. Tris–HCl buffer is used to stabilize Sb# 14.

#### *4.1.3 Reducing agents*

To reduce aggregation, reducing agents such as dithiothreitol (DTT) may be used and added to the buffer. DTT is called Cleland's reagent and is used for protein reduction. However, a high concentration of DTT can reduce the nickel ion in the resin of the column. That is why the determination of the optimal concentration of DTT is essential. β-ME (Beta-mercaptoethanol) cleaves protein disulfide bonds (cystine), and TCEP (Tris phosphine hydrochloride) can also be used as reducing agents, considering longer half-life β-ME. DTT reacts easily with nickel ions whereas β-ME reacts easily with cobalt, copper ions, and other phosphate buffers [44]. A precaution is required to obtain optimal conditions.

#### *4.1.4 Isoelectric point (pI) and pH*

Each protein has a pI, where the protein's net charge is zero. Protein does not migrate at that point, and aggregation occurs [45]. On one hand, acidic proteins are likely to crystallize 0–2.5 pH units above their isoelectric point. On the other hand, basic proteins are more likely to crystallize 1.5–3 pH units below their pI. Hence, different pH values affect the protein's stability and solubility [46]. The pI of the peptide is important for us to know the pH range where that peptide has a negative charge and to prepare the buffer solution accordingly. That is why the pH of the buffer component is one of the most critical parameters. Sb #14 has a pI value of 8.91. This value set the pH parameter (pH:7.91) of the buffer used in the purification process.

#### **4.2 Importance of protein isoelectric point in tagged protein purification**

pI represents the pH level of a molecule where the net charge is zero. Amino acid composition of the protein can be used to calculate an estimated value with the help of databases [47]. If the pI is lower than the pH of a solution, protein will have a negative charge but if it is the opposite then the protein will have a positive charge. This feature can be used for purification purposes since it is a specific physicochemical parameter to distinguish between amphoteric molecules [39]. Also, it can be used to understand how solution pH can affect the protein stability in the pH range. So, buffers are used to keep proteins stable. To create an environment for protein to be stable, generally, the buffer is selected to have a pH level around the pI of the protein. If this difference between the pI of protein and pH of the solution gets larger then protein gets a greater net charge too. With this greater net charge, ionic compounds will be able to bind residues [48]. To avoid this unspecific interaction, the buffer's pH range should be selected accordingly to the protein's pI. And this knowledge of pH values with their effects on the proteins can be useful in the purification process. In tagged protein purification, affinity chromatography is a commonly used technique. The pH levels also affect this technique since affinity resins have their pH ranges to provide more stable links for not only the ligand and the bead but also for the tag and the ligand. While making decisions about the purification protocol, the affinity resins' and the tags' working pH ranges should be kept in mind to create a better environment and more stable interaction. Also choosing affinity resins and tags that have a wider range of pH that they can work may be useful for the purification of proteins.

#### **4.3 Protein aggregation and importance of ionic strength**

Proteins are special structures that work with covalent and non-covalent interactions. They have cellular wide roles, including signaling, structural and metabolic processes. Their special structural features and 3D architecture determine their roles and interactions. These forms of proteins are determined by the amino acid sequences [49]. Proteins are not synthesized in their functional form. When their translation process is finished, the primary protein structure is formed. After that, they form alpha helixes and beta sheets by hydrogen bonds. Alpha helixes and beta sheets interact with each other with weak interactions and disulfide bonds and tertiary structure is formed. In some instances, the quaternary structure may be formed when tertiary structures interact and eventually in all cases functional protein forms [55]. However, in some cases, proteins can accumulate and form aggregates which may cause failure in protein purification experiments. Mostly, beta-sheets tend to interact with each other and accumulate. This event can be exampled by amyloid aggregates in Alzheimer's disease. Recombinant protein aggregates resulted in the prevention of exposure of tags in tagged protein that causes failure in purification. Also, solubility prevents aggregate formation in proteins [50]. Protein aggregation can be prevented by adding salt to the proteins. Salt ions interact with the charged protein surface areas and prevent non-specific interactions, aggregation, and lower protein–protein interaction. However, a precaution is a must when preparing protein for binding experiments. Please note that high ionic concentration blocks ligand/protein binding experiments. As shown in **Figure 2**, Sb #14 is prone to aggregation and the process may be prevented/decreased through proper conditions.

*Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

#### **Figure 2.**

*Ionic strength is important for the separation of proteins from each other that can be aggregated. The strength of ions resists accumulation by preventing protein-protein interaction.*

#### **4.4 Usage of additional agents to prevent protein accumulation, attachment, and insolubility**

Urea dissolves the aggregated protein solutions. The efficiency of the process is increased by taking the necessary purification steps [1, 51]. Among these processes, protein dissolving and refolding steps constitute the most important steps for optimal protein activity and higher recovery. The protein precipitate is generally separated from other cellular components by low-speed centrifugation after cell lysis. Because protein aggregates are denser than cellular components, the lysate proteins are precipitated by centrifugation and then dissolved using detergents such as urea, guanidine-HCl, high concentrations of chaotropic denaturants, sodium N-lauroyl sarcosine, SDS, N-acetyl trimethyl ammonium chloride [52]. Further, additional reducing agents such as DTT, cysteine, Triton X-100, β-ME are used to dissolve inclusion bodies. These agents retain cysteine residues, minimizing the formation of false and unnatural disulfide bonds in the protein solution. Metal-containing oxidation of cysteine is prevented by using chelating agents such as EDTA in dissolution buffers [44, 52]. By removing the soluble protein content, removing the chaotropic reagents, and diluting them directly into the renaturation buffer, the recombinant

proteins are folded back into their native form [44]. Protein collapse is a higher-order reaction while protein folding is a lower-order reaction. Therefore, the aggregation rate is higher than the folding rate. Due to the kinetic competition that occurs, the increase in protein concentration decreases the folding efficiency of the protein. For accurate and efficient folding kinetics, the preferred protein concentration is used in the range of 10–50 μg.ml−1 [1, 53]. As explained in the section of 'Disulfide bond formation', recombinant proteins with multiple disulfide bonds in their structure tend to be in a correct folding process in the presence of both oxidizing and reducing agents for the formation of these bonds. The simplest way for oxidation is to oxidize the protein with air in the presence of a metal catalyst. Another common oxidation option is the addition of thiol agents containing compounds such as glutathione, cysteine, cysteamine to the protein mixture. The most commonly used thiol reagents are reduced/oxidized glutathione (GSH-GSSH), cysteine/cystine, DTT/GSSH, cysteamine compounds [1, 36]. There are also low-molecular-weight additives that help refolding process. There are studies on the use of additives such as acetone, DMSO, short-chain alcohols, PEG in the bioactive protein process. In addition, it has been observed that L-arginine/HCl reduces aggregation on protein. The 0.4–1 M arginine used in the studies also increases the protein folding efficiency by reducing the aggregation in the recombinant protein solution. This feature of arginine has been attributed to the interaction of the guanidino structure in its structure with tryptophan residues in proteins [44, 52–55]. Sb #14 was also treated with DTT but when overexpressed, the protein has solubility problems. The structure is highly prone to aggregation and solubility may be increased upon co-expressing chaperones/Heat Shock Proteins or yeast systems seem proper for preventing aggregation. Yet, this may not solve the problem but mutational studies may provide more soluble and stable structures.

#### **5. Conclusion**

Protein purification depends on several factors: resin type, solvent, ionic strength, pH, protein structural tendency to aggregation, buffer systems, protein structure, ligand if any, column dimension. For each factor, problems may be encountered. To eliminate these problems and decide on protein purification protocol, protein structural properties must be examined initially. Tandem purification steps may also increase the purification yield. However, self-cleavage of certain proteins or oxidation that may distort the protein function leads to problems. Therefore, several distinct protocols may be tested before purifying the targeted protein with high efficiency and functionality. Sb#14 structure mainly consists of β-sheets and overexpressing this petit protein lead aggregation. Solubility is another problem in the cellular milieu as Sb#14 hydrophobic nature interacts with other proteins in the lysate. Therefore, proper solvent selection (phosphate buffer) and adjusting the pH (1 unit lower than pI, 7.91) provide soluble protein. Further, we take advantage of the protein's lower molecular weight and employed a convenient resin (Superdex 75-separates 3000–70,000 molecular weights, most of the lysate elutes before Sb#14) and custom size column (15 cm length with r: 3 cm-lower pressure yet increase resolution). The purification was performed with high yield by AKTA go FPLC system. Additionally, co-expressing heat shock proteins with this type of protein may help in folding and dissolving aggregates. All these conditions must be tested for individual proteins for optimum purification yield.

*Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial… DOI: http://dx.doi.org/10.5772/intechopen.103958*

#### **Acknowledgements**

Prof. Dr. Yusuf TUTAR acknowledges grant from TUSEB (Project # 8970-220-CV-01) and infrastructure grant from the University of Health Sciences-Turkey (Project #2017-041). Sb#14 is from Addgene (#153522).

### **Author details**

Kubra Acikalin Coskun1 , Nazlıcan Yurekli<sup>2</sup> , Elif Cansu Abay2 , Merve Tutar3 , Mervenur Al4 and Yusuf Tutar5,6,7\*

1 Faculty of Medicine, Division of Medicinal Biology, Department of Basic Sciences, Istanbul Aydin University, Istanbul, Turkey

2 Department of Molecular Biology and Genetics, Arel University, Istanbul, Turkey

3 Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

4 Hamidiye Faculty of Medicine, Division of Medicinal Biochemistry, Department of Basic Sciences, University of Health Sciences-Turkey, Istanbul, Turkey

5 Hamidiye Faculty of Pharmacy, Division of Biochemistry, Department of Basic Pharmaceutical Sciences, University of Health Sciences-Turkey, Istanbul, Turkey

6 Division of Molecular Oncology, Hamidiye Health Sciences Institutes, University of Health Sciences-Turkey, Istanbul, Turkey

7 Validebağ Research Center, University of Health Sciences-Turkey, Istanbul, Turkey

\*Address all correspondence to: yusuf.tutar@sbu.edu.tr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 2**

## Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative Diseases and Metabolic Disorders

*Ohanube A.K. Goodluck, Obeta M. Uchejeso and Ikeagwulonu R. Chinaza*

#### **Abstract**

An accurate diagnosis gives leeway to cost-effective treatments. However, many diseases continue to evolve; hence, their etiology is sometimes missed due to the procedures used during diagnosis. Protein-related diseases include proteopathies (proteinopathies) such as neurodegenerative diseases and metabolic disorders like protein-energy malnutrition and some hormonopathies. Hormonopathies are associated with the change in the production of hormones. Diabetes mellitus, a type of hormonopathy, is reviewed in this work alongside neurodegenerative diseases and protein-energy malnutrition. This chapter aims to elucidate more on the diagnosis of these diseases considering the structure and function of their proteins viz-a-viz their deficiencies and hyper-production in man. Their pathogenesis and the principles underlying their diagnosis are further discussed to optimize the management of these diseases among patients.

**Keywords:** prion, proteinopathy, hormonopathy, marasmus, kwashiorkor, alzheimer, parkinson, huntington, diabetes, neurodegenerative disease

#### **1. Introduction**

Medical laboratory diagnosis has given hope to detect and efficiently treat or manage diseases, and protein-related diseases are not an exception. The diagnosis ranges from the least like urinalysis to the highly sophisticated methodologies involving molecular techniques such as polymerase chain reaction. This chapter discusses the neurodegenerative disease and the pathologic conditions associated with proteinderived hormones and protein-energy malnutrition, focusing on their diagnosis and management.

Proteinopathy (proteopathy) is a disease mainly characterized by the production of aberrant proteins. Here, there could be a defect in the structure or function of the proteins produced, which could also reflect in the over-secretion or under-secretion

of these proteins. Neurodegeneration involves a gradual loss of neuronal structure or function, invariably leading to cellular death [1]. Neurodegenerative proteinopathies (proteopathies) are neurodegenerative diseases that have abnormally produced proteins that could result from the changes in the structure and function of these proteins. Their pathological examination reveals their link with aberrant proteins. Their prevalence in the United States of America is shown in **Table 1**. Popular


#### **Table 1.**

*Prevalence of some common neurodegenerative diseases in the United States [1–5].*


*PrP, prion protein; PrPc , cellular prion protein; PrPsc, scrapie isoform of the prion protein; CJD, Creutzfeldt–Jacob disease.*

#### **Table 2.**

*Pathogenic feature of the types of prion disease [1, 2].*

*Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*


#### **Table 3.**

*Protein detection in prevalent neurodegenerative diseases.*

examples of neurodegenerative diseases are amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease, which may be grouped as non-infectious neurodegenerative proteinopathies, and prion diseases considered infectious neurodegenerative proteinopathies [1, 2]. The pathogenic feature of the variety of prion disease, an infectious neurodegenerative proteinopathy, is shown in **Table 2**, while protein deposition seen in most common neurodegenerative diseases is shown in **Table 3**.

#### **2. Infectious neurodegenerative proteinopathies**

#### **2.1 Prion diseases**

Infectious neurodegenerative proteinopathies are proteinopathies that are communicable diseases, and their characteristic symptom is neurodegeneration in humans. Prion disease is an excellent example of such a disease. Some types of prion disease, also known as transmissible spongiform encephalopathies (TSEs), such as Creutzfeldt–Jakob disease (CJD), Kuru, Gerstmann–Straussler–Scheinker syndrome (GSS), and fatal familial insomnia, are a group of fatal neurodegenerative diseases. They are incurable but manageable; they can affect humans and animals and are sometimes transmitted to humans by infected meat products. The clinical progression is over weeks, progressing to akinetic mutism with a median disease duration of 20 weeks. Prodromal features, present in around 30% of cases, include depression, fatigue, weight loss, headaches, insomnia, general malaise, and ill-defined pain sensations. Additionally, we see myoclonus and mental deterioration, while neurological features include pyramidal signs, extrapyramidal signs, cerebellar ataxia, and cortical blindness [13–15]. The pathogenic features of the prion disease are shown in **Table 2**. Risk factors for prion disease are positive family history, eating meat infected by "mad cow disease," infection from receiving contaminated organs or tissues including corneal tissue, or contaminated medical equipment [16].

The central feature of the pathogenesis seen in most types of prion disease is the post-translational conversion of host-encoded, normal, healthy, cellular prion protein (PrPC) to an abnormal infectious isoform, termed scrapie isoform of the prion protein (PrPSc) or (PrPres), which is an alternatively folded variant of the cellular prion protein, PrPC [15–17]. This misfolding of PrPSc is possible due to the more important content of the β-sheet structure, which aggregates to form medium and large-size polymers [18]. Studies have proposed different functions of PrPC, such as the roles in apoptosis, neuroprotection, oxidative stress, transmembrane signaling, cell adhesion, myelination, and trafficking of metal ions. The critical event in the stages of prion disease is the structural and conformational change of PrPC to the disease-associated misfolded form, PrPSc [18]. This conversion changes PrPC from a protein characterized by alpha-helices to a partially protease-resistant misfolded protein filled with beta-sheets (β-sheets). Proteinase K (PK) partially digests PrPSc and is often used to determine the presence of misfolded PrPSc. PrPSc accumulates in different brain regions as distinct types of deposits depending on the animal species and strains of the infectious agent. The incapacitation of the critical biological function of PrP<sup>C</sup> is one possible mechanism by which PrPSc formation might lead to degeneration of neurons. Another possible mechanism by which PrPSc formation might be linked to the disease is by direct toxicity of the misfolded protein [15–18]. **Table 2** shows the list of prion diseases and their routes of infection. Prion disease affecting animals has been included for academic purposes; however, our discussion is focused on those affecting humans.

#### **2.2 Etiology and clinical manifestation**

Human prion diseases can be grouped etiologically into sporadic, inherited, and acquired forms [13]. The following paragraphs shall consider the different etiological classifications of prion diseases, stating the respective examples.

#### *2.2.1 Sporadic cases*

More than 80% of the occurrence is sporadic cases of human prion disease, which presents as Creutzfeldt–Jakob disease (sporadic CJD). The cause of sporadic CJD is unknown, although it is hypothesized to include somatic mutation of the prion protein gene (*PRNP*) or the spontaneous conversion to PrPSc form of PrPC. A polymorphism that occurs at residue 129 of human PrP (encoding either methionine (M) or valine (V)) strongly influences the susceptibility to human prion diseases. About a third of Europeans are homozygous for the more frequent methionine allele, half are heterozygous, and a tenth is homozygous for valine. Homozygosity at *PRNP* codon 129 is a causal factor to the development of sporadic and acquired CJD. Polymorphic homozygosity favors the occurrence of most sporadic CJD. This susceptibility factor is also vital in the inherited forms of CJD, most especially in vCJD. All hospitalized cases studied so far have been homozygous for codon 129 methionine of *PRNP*. Additionally, a haplotype for *PRNP* susceptibility has been identified, indicating additional genetic susceptibility to sporadic CJD at or near the *PRNP* locus [15–18].

Classical sporadic CJD presents with a rapidly progressive multifocal dementia predominantly with myoclonus. The onset is usually in the 45–75 years age group, with the median age at death of 68 years. The clinical progression expends over weeks, progressing to akinetic mutism with a median disease duration of 5 months. Prodromal features, present in about 30% of cases, include fatigue, insomnia,

#### *Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

headaches, weight loss, depression, malaise, and non-specific pain sensations. In addition to mental deterioration and myoclonus with cerebellar ataxia, frequent additional neurological features include extrapyramidal signs, pyramidal signs, and cortical blindness [13, 18].

Atypical forms of sporadic CJD are seen in about 10% of cases of CJD, and they have a longer duration of a clinical course spanning over 2 years. Here, cerebellar ataxia is seen instead of cognitive impairment. Hence, it is termed ataxic CJD [19]. Heidenhain's variant of CJD refers to conditions in which cortical blindness is marked with severe involvement of the occipital lobes. The panencephalopathic type of CJD is more common in Japan; it presents with extensive degeneration of the cerebral white matter and spongiform vacuolation of the gray matter [20].

#### *2.2.2 Inherited prion disease*

About a fifth of human prion diseases is associated with autosomal dominant pathogenic mutations in *PRNP* [21–23]. The mechanism by which pathogenic mutations in *PRNP* cause prion disease is yet to be elucidated; however, it is believed that in most cases, it involves a mutation that leads to an increased tendency of PrPC to form PrPSc. Even though pieces of evidence abound in congruence with this, this may partly be related to the decreased thermodynamic stability of mutated PrPC [24, 25].

Traditionally, inherited prion diseases have been classified by the presenting clinical syndrome, falling into three main sub-divisions: GSS, CJD, or FFI. GSS is seen in people in their 40s; it classically presents as chronic cerebellar ataxia with pyramidal features with dementia seen much later in a clinical course that is usually longer than in classical CJD [13, 23]. Fatal familial insomnia (FFI) has its pathognomonic feature as progressive chronic insomnia, dementia and dysautonomia, selective thalamic degeneration, and is mainly associated with a missense mutation at codon 178 of *PRNP* (3); its sporadic form with no causative mutation in *PRNP* have been reported [13, 23, 24]. Another form of inherited prion disease, though extremely rare, is variably protease-sensitive prionopathy (VPSPr). VPSPr is similar to CJD; however, the protein is less sensitive to digestion. It is more likely to affect people in their seventh decade of life with a family history of dementia. The existence of phenotypic overlap between individuals with different mutations and even in family members with the same *PRNP* mutation indicates that accurate classification of inherited human prion diseases should be based upon mutation alone [24–26]. Due to the extensive phenotypic variability associated with inherited prion disease and its ability to mimic other neurodegenerative conditions, notably Alzheimer's disease, *PRNP* analysis should be considered in all patients with undiagnosed dementing ataxic disorders [13, 23, 26].

#### *2.2.3 Acquired prion disease*

Human prion diseases are transmissible diseases; their acquired forms have, however, until recently, been confined to rare and unusual situations. They include the iatrogenic CJD, Kuru, and variant CJD.

The two most prevalent causes of iatrogenic CJD occurring through the medical procedure are the implantation of grafts of dura mater and treatment with growth hormone derived from the pituitary glands of human cadavers [13]. Less frequent causes of human prion disease have been associated with the iatrogenic transmission of CJD during corneal transplantation, infected electroencephalographic (EEG) electrode implantation, and surgical operations using contaminated instruments or

apparatus [13, 27, 28]. The clinical presentation in iatrogenic forms of human prion disease appears to be related to their etiology and, in particular, the route of exposure to human prions [13]. Peripheral routes of infection are commonly associated with more extended incubation periods and usually present with a Kuru-like syndrome, in which ataxia is common, while dementia is rare at the onset. Conversely, patients with dura mater graft-related exposure to human prions, in which infectivity is placed proximal to the brain, usually have a clinical presentation that looks like sporadic CJD, although exceptions with unusual clinical features have been reported [13, 27–29].

Kuru is a disease that used to be predominant among cannibals in the Fore tribe of the Eastern Highlands in Papua New Guinea, but it is now rare due to consistent enlightenment and rules that abolished such culture [29, 30]. It is caused by eating prion ladened human brain tissue. The central clinical feature of Kuru is progressive cerebellar ataxia, and in sharp contrast to sporadic CJD, dementia is late and may be absent. A prodrome and three clinical stages consisting of an ambulatory stage, a sedentary stage, and a tertiary stage have been described [13, 23, 29]. Remarkably, Kuru demonstrates that incubation periods of infection with human prions can exceed 50 years [29]. The *PRNP* codon 129 genotype has been identified to have a pronounced effect on Kuru in terms of the incubation periods and susceptibility, and most elderly survivors of the kuru epidemic are heterozygotes [26, 30, 31]. The glaring survival advantage for codon 129 heterozygotes gives a cue for a robust basis for selection pressure in the Fore clan [13, 23, 26]. However, analyzing the global haplotype diversity and frequency of the alleles responsible for coding and noncoding polymorphisms of *PRNP*, an older and widely spread balancing selection at this locus has more unusual variation because of heterozygote advantage is suggestive [23, 29, 31]. Only a few human genes present evidence for balancing selection. With the biochemical and physical evidence of cannibalism on five continents, one explanation is that cannibalism resulted in prion disease epidemics in human prehistory, thus imposing balancing selection on *PRNP* [23, 29, 31].

The variant CJD is an infectious type of disease that is related to "mad cow disease." Eating meat that has been inflicted with bovine spongiform encephalopathy (BSE) may cause the disease in humans, as seen in the United Kingdom years back [13, 27]. The meat may cause abnormal development of normal human prion protein. The disease is associated with iatrogenic conditions. This disease usually affects younger people and is rare in most developed nations [13, 27, 32].

#### **2.3 Diagnosis of prion**

Prion disease can be provisionally diagnosed using the clinical signs and symptoms presented alongside the taking of history. Neurologic and visual examinations could be done to ascertain nerve damage and vision loss. Prion diseases such as CJD can be diagnosed via MRI, PET, and CT scans of the brain and body; and spinal tapped cerebrospinal fluid (CSF). Electroencephalogram, which analyses brain waves, could also be used; this painless test requires placing electrodes on the scalp. At the same time, some centers choose to do blood and urine tests, which involves immunologically based analysis. Raised cerebrospinal fluid 14-3-3 protein, S-100, and neuronal-specific enolase (NSE), although unspecific for CJD, may be helpful diagnostically in the appropriate clinical context [13].

Prions lack DNA or RNA, so PCR or other nucleic acid-based tests cannot identify them. Hence, the strategy is to mix the test material with the proteinase K (PK)

*Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

enzyme, which digests the regular portion of prion protein but cannot digest any of the portions, which appears abnormal. Some other techniques aim at detecting the residual protein (PrPSc) after digestion. Methods relying on PK digestion are less sensitive than those that do not rely on it because the former reduces the small amount of original PrPSc captured [33].

The most sensitive, crucial, precise, but uncommon immunoassay method of confirmatory diagnosis is by identifying the disease-causing PrP isoform (PrPSc) using the conformation-dependent immunoassay (CDI) laboratory method [15, 34, 35]. The CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc [14]. The CDI was developed to quantify PrPSc in tissue samples from mammals producing prions. Sandwich CDI represents a rapid, robust, powerful tool to study prions in bodily fluids of CJD/vCJD patients, with a turnaround time of 12–24 hours [15, 34]. Safar et al., in their experiment, showed the superior performance of the CDI in diagnosing prion disease compared to the routine neuropathologic examination and immunohistochemistry (IHC). Hence, they proposed using CDI in place of these earlier mentioned methods [14, 33].

#### **2.4 Managing prion diseases**

Prion diseases rarely have a cure; hence they are managed using certain medications, which could slow their progress. This management focuses on keeping people with these diseases as safe and comfortable as possible despite progressive and debilitating symptoms.

Effective anti-prion agents may have broader implications due to the adverse effects associated with them. Several therapeutic approaches include polyanionic, polycyclic drugs such as pentosan polysulfate (PPS), which prevent the conversion of PrPc to PrPres and might also sequester and down-regulate the protease-resistant prion protein (PrPres). Polyanionic compounds might also help to clear PrPres. Treatments aimed at the laminin receptor, an essential accessory molecule in converting PrPc to PrPres—neuroprotection, immunotherapy, siRNA, and antisense approaches, have provided some experimental cues [28].

In drug development, the PrPC, PrPSc (PrPres), or the process of its conversion are the targets. Pentosan polysulphate (PPS) is presumed to act as a coreceptor for PrP on the cell surface in competition with endogenous heparin sulfate proteoglycans and shows the ability to inhibit the formation of new PrPSc in neuroblastoma cells. Quinacrine is thought to prevent PrPSc polymerization by stabilizing PrPC and reducing its conversion to PrPSc. Doxycycline reverses the protease resistance of PrPSc extracted from CJD brains and prolongs the survival of animals experimentally infected with prions, even when given at the onset of clinical signs [33, 36–38]. Active and passive immunization are two significant aspects of immunotherapy. Resveratrol is an essential compound with antioxidant, anti-allergy, anti-aging, and neuroprotective activities, and it has been reported to eliminate prion replication *in vitro* and prion infection *in vivo*. The ubiquitin (Ub)-proteasome system (UPS) is the first line of defense in degrading soluble misfolded proteins. Conversion from PrPC into PrPSc may involve chaperones and Ub ligases for UPS-dependent protein quality control. Enhanced UPS aims to stimulate the degradation of PrPSc. The autophagy-lysosome system is another quality control system to remove the misfolded proteins [36–38]. Studies have alluded that rapamycin can activate autophagy *in vitro* and delay disease onset in rodents with prion disease [39]. Autophagy could also lead to PrPSc clearance in cell models and prolong the lifespan of prion-infected mice [36].

#### **3. Non-infectious neurodegenerative proteinopathies**

#### **3.1 Alzheimer disease**

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the leading cause of dementia among geriatrics [6, 40]. It affects over 27 million persons worldwide, and prediction shows that over 86 million people would be affected by 2050 [7]. It is characterized by difficulty solving problems, memory loss, disorientation in time and space, among others [41]. This disease was first described in 1906 at a conference in Tubingen, Germany, by Alois Alzheimer [41]. Aging seen in the absence or presence of dementia of the Alzheimer type (DAT) is associated with loss of weight; hence, accelerated weight loss of idiopathic origin may herald the onset of DAT, aiding its clinical diagnosis [42]. The significant risk factors of this multifactorial disease include apolipoprotein E 4, hypercholesterolemia, genotype, traumatic brain injury, family history, age, obesity, hypertension, diabetes, and low level of education [6].

A complex array of molecular events has been implicated in the pathogenesis of AD. The major pathological characteristics of AD brains are senile, neurofibrillary tangles, plaques, and neuronal loss [6, 7, 40, 41]. The pathogenic mechanism implicated here seems elusive; however, oxidative stress has been identified as a leading factor in the initiation and progression of the ailment [43]. The excessive reactive oxygen species may be generated from mitochondria dysfunction and aberrant accumulation of transition metals, while the abnormal accumulation of amyloid-beta (Aβ) and tau proteins appears to promote the redox imbalance leading to neurotoxicity [41–43]. Additionally, oxidative stress may augment the production and aggregation of Aβ and facilitate the phosphorylation and polymerization of tau, leading to a vicious cycle that promotes the initiation and progression of AD [43]. Researches are gradually drifting from the simple assumption of the original amyloid hypothesis to new theories of pathogenesis, which include gamma oscillations, cerebral vasoconstriction, prion transmission, growth hormone secretagogue receptor 1α (GHSR1α) mediated mechanism, and infection [44].

#### *3.1.1 Diagnosis*

The disease morphologically features an overall loss of synapses and neurons and an overall reduction in brain volume. The neuropathologic examination has been identified as the gold standard for diagnosing Alzheimer's disease (AD). However, popular opinion has it that histologic examination is the best indicator of AD diagnosis. Thus, an autopsy may gradually become the gold standard for determining clinical diagnostic accuracy rates [7, 45, 46]. A routine examination is better done with magnetic resonance (MR) or computed tomographic (CT) imaging. In the early onset of the disease, coronal MR images have been helpful to document or quantify the atrophy of both the hippocampus and entorhinal cortex. At the same time, subtraction and volumetric MR techniques can be used to quantify and monitor rates of regional atrophy and dementia progression. Positron emission tomography (PET) coupled with single-photon emission CT is helpful in the differential diagnosis of AD from other dementias associated with the cortical and subcortical dementias and may also be of prognostic value. Values from the MR are also used to monitor treatment effects in clinical trials of antidementia agents and cognitive enhancers [7, 40, 46].

Additionally, PET studies have shown that subtle abnormalities may occur at the prodromal stages of AD and in subjects bearing susceptibility genes. PET ligands

#### *Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

may be of value in identifying amyloid plaques. Functional MR-based memory challenge tests are also beneficial [7, 40, 46].

Peripheral biomarkers are also beginning to gain ground in the diagnosis of Alzheimer's disease. This gives room for presymptomatic detection of disease, which could be valuable for monitoring the efficacy of disease interventions during clinical trials. CSF has long remained the sample of choice for biomarkers for many scientists until some Australian scientists developed theirs using blood. A biomarker panel that was about 85% sensitive and 93% specific was developed. The plasma markers in this biomarker panel that was significantly increased were cortisol, pancreatic polypeptide, β2 microglobulin, insulin-like growth factor binding protein 2, and vascular cell adhesion molecule 1. There was also CD40, carcinoembryonic antigen, matrix metalloprotein 2, macrophage inflammatory protein 1α, superoxide dismutase, and homocysteine. In AD, these markers were decreased (apolipoprotein E, epidermal growth factor receptor, hemoglobin, calcium, zinc, interleukin 17, and albumin). This panel of plasma biomarkers was proven to be efficient, as it distinguished individuals with AD from cognitively healthy control subjects with high precision [7, 46].

Nevertheless, the prominent causal factors for AD development are genetic mutation involving genes encoding for proteins such as presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Usually, at an early age between the third and fifth decade of life, about half of the carriers of such mutations develop AD-type dementia. The hallmark of AD includes the accumulation of Aβ as senile plaques and aggregating hyperphosphorylated tau-mediated neurofibrillary tangles, NFTs for short [6, 7].

#### *3.1.2 Management*

Previous researches implicated an instability in the homeostasis of neuronal Ca2+ in age-related cognitive impairment associated with Alzheimer's disease (AD). This is seen when increased oxidative stress and impaired energy metabolism associated with senescent neurons lead to malfunctioning proteins that control membrane excitability and subcellular Ca2+ dynamics. Toxic forms of amyloid β-peptide (Aβ) may trigger Ca2+ influx into neurons by inducing membrane-associated oxidative stress or forming an oligomeric pore in the membrane, thus, exposing neurons to excitotoxicity and apoptosis. During AD, mutations in the β-amyloid precursor protein and presenilins may compromise the normal proteins in the plasma membrane and endoplasmic reticulum. With time, knowledge of the actions of Ca2+ upstream and downstream of Aβ gave a cue to developing some prophylactic or curative interventions for AD [47].

Alzheimer's disease is managed, not cured; the only medications approved for managing the disease are used for mild to moderate AD. These drugs are the cholinesterase inhibitors (ChEI): tacrine, rivastigmine, donepezil, and galantamine. While for moderate to severe AD is memantine, a noncompetitive *N*-methyl-d-aspartate (NMDA) receptor inhibitor, which blocks the excess release of glutamate assumed to be related to cholinergic damage [6, 48, 49].

Research has shown no additional benefit of combination therapy involving vitamin E (2000 IU/day) and selegiline (10 mg/day). However, due to low cost and relative safety, vitamin E was recommended in addition to ChEIs to slow AD progression. Some other treatments, such as Ginkgo biloba, anti-inflammatory drugs, and hormone replacement therapy, have been suggested as possible treatments, although with insufficient evidence [6, 48, 49].

Another treatment option is a combination of memantine and donepezil, and the combination therapy (Namzaric®) was recommended in 2014 to treat individuals with moderate to severe AD who are stabilized on donepezil and memantine therapy. The multi-target-directed ligands (MTDLs) approach currently focuses on designing hybrid molecules that simultaneously regulate multiple biological targets. Moquin is a drug, which has been developed as a potential anti-AD candidate because of its MTDL design capacity. However, combination therapy (CT), including ChEIs and memantine, currently constitutes the best and effective treatment for individuals displaying moderate-to-severe AD. Additionally, CT exhibited better clinical efficacy than monotherapy, along with similar tolerability and safety [6].

#### **3.2 Parkinson disease**

Parkinson's disease (PD) is a neurodegenerative clinical syndrome characterized by at least two of four cardinal features: bradykinesia, rigidity, resting tremor, and impairment of postural balance leading to disturbance of gait and falling. James Parkinson, an English physician, was the first to describe this disease, when he called it the "shaking palsy" in 1817 and also coined the term, *paralysis agitans* meaning the shaking palsy, since then, there is still a lack of understanding of the causes of PD [8, 50]. Parkinson's disease may be mistaken with the regular essential tremor; however, the difference in both is that the tremor in Parkinsonism occurs predominantly at rest, while that of essential tremor is seen during actions. Also, tremor in Parkinsonism is unilaterally seen in the arms or legs, while essential tremor bilaterally affects both upper limbs. Bradykinesia is usually the most troublesome symptom. Patients report slowness in performing their daily activities. Falls and swallowing problems are classic signs of late Parkinson's disease; however, if they occur early and are accompanied by unresponsiveness to treatment, they may indicate multiple system atrophy or progressive supranuclear palsy. Early dementia and other features could indicate Lewy body dementia, vascular Parkinsonism, or corticobasal degeneration. Young patients with Parkinsonism (aged <40 years) should always be evaluated for changes in the values of their serum copper and ceruloplasmin levels, with a 24-hour urine collection for copper excretion and slit-lamp examination for Kayser–Fleischer rings in consideration of Wilson's disease [51].

The pathognomonic feature of PD is a loss of the pigmented, dopaminergic neurons of the substantia nigra pars compacta in the brain, with the appearance of intracellular inclusions known as Lewy bodies. During the 1960s, researchers identified a fundamental defect that is a hallmark of the disease: the loss of brain cells that produce an essential chemical, dopamine, which helps direct muscle activity. Gradual loss of dopamine-containing neurons is a feature of normal aging; however, most people do not lose 70–80% of the dopaminergic neurons that cause symptomatic PD. In the absence of treatment, PD gradually deteriorates into a rigid, akinetic state where patients cannot care for themselves within 5–10 years. Death may result from complications of immobility, such as aspiration pneumonia and pulmonary embolism [50, 51].

#### *3.2.1 Diagnosis*

This aims to identify ubiquitous Lewy bodies in microscopic postmortem studies, a feature of cell death associated with the disease. However, the clinical diagnosis in PD includes cardinal motor symptoms such as akinesia, rigidity, and tremor [8].

#### *Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

Diagnosis of Parkinsonism involves structured clinical examinations or autopsies. The central pathology in PD is the degeneration of pigmented neurons in the brainstem. Through a microscope, intracellular Lewy bodies are easily identified. The neurons located in the substantia nigra pars compacta are the most affected, resulting in dopamine depletion to its major projection area, the striatum. The depletion culminates into an overactive subthalamic nucleus, which increases the activity of the major inhibitory output nuclei such as the globus pallidus and substantia nigra pars reticulata, resulting in increased inhibition of thalamic activity and problems with motor output. Lewy bodies in the cortex and deeper structures are the main features that distinguish Parkinson's disease (PD) from dementia with Lewy bodies (DLB), a type of neurodegeneration sharing similarities with Alzheimer's and Parkinson's [9].

Due to the presence of Lewy-type α-synucleinopathy in the submandibular glands of PD patients, some scientists considered the feasibility of submandibular gland biopsy for diagnosing PD. Hence, immunohistochemical staining was considered for Lewy-type α-synucleinopathy [10]. Some studies also considered performing needle core biopsies of the submandibular gland in living patients with PD to assess Lewytype α-synucleinopathy (LTS). Although it was a small-scale study, this tissue biopsy method may be valuable for confirming PD in patients being considered for invasive medical interventions and research studies of other PD biomarkers [52].

Autopsy remains the main definitive diagnostic tool. Some studies provided evidence that unilateral onset of symptoms with features that include tremor and at least one of bradykinesia and rigidity with an efficient initial response to l-dopa have been the best predictors of the pathological diagnosis. In a fifth of the cases, a different neurological disorder was diagnosed at autopsy from that diagnosed during life. Neurological imaging studies with computed tomography or MRI do not reveal any specific changes related to Parkinson's disease. However, most neurologists perform brain imaging tests to rule out rare conditions requiring a different treatment regimen and management strategies, such as normal pressure hydrocephalus or focal lesions. Functional imaging of brain regions affected by Parkinsonism with either positron emission tomography (PET) or single-photon emission tomography SPECT has been proposed [51].

#### *3.2.2 Management*

For the efficient management of medical conditions, the risk–benefit ratio is considered; the aim here is to make the patient experience wellness as close to normal function as possible without having side effects from therapy. Hence, the appropriate multi-disciplinary approach must be utilized.

Some factors are considered to determine the optimal choice for the individual patient at different phases. These include the following:


Pharmacological attempts to restore dopaminergic activity with levodopa and dopamine agonists have successfully alleviated many of the clinical features of PD. A complementary approach has been to resuscitate the normal balance of cholinergic and dopaminergic influences on the basal ganglia with anticholinergic drugs. The availability of effective pharmacological treatment has drastically altered the prognosis of PD; in many cases, good functional mobility can be achieved for many years, and the life expectancy of well-managed patients is increased substantially [50]. It is important to emphasize that PD therapy must be individualized and tailored to the specific needs of each patient using a basic algorithm [53, 54].

Treatment of early PD with mild symptoms benefit from nonpharmacological therapy such as exercise and relaxation techniques. However, monoamine (MAO) inhibitors such as selegiline, rasagiline, and safinamide; dopamine agonists; or anticholinergic medications are added to ameliorate conditions [50, 53]. Levodopa or dopamine agonist could be added to ease the challenges associated with the motor neurons [50, 53]. Decades of clinical observation have validated levodopa as the most effective primary medicinal agent [50]. Entacapone (Comtan) and rasagiline (Agilect) could help hold brief pending when PD has progressed and the medications seem inefficient in relieving symptoms [50]. Surgical and experimental therapeutics should be considered as the disease progresses and motor complications (including motor fluctuations and dyskinesias) develop [53]. Inosine, which increases urates, and Isradipine, a calcium channel blocker, when added, are treatments designed to prevent the accumulation of toxic α-synuclein. Monoclonal antibodies directed at aggregated α-synuclein in some patients with Parkinson's disease also provided evidence of strong target engagement and CNS penetration [53].

Optimizing the pharmacologic treatment for both motor and non-motor symptoms is critical; however, nutritional interventions cum counseling could also be planned to manage weight gain or loss of weight efficiently. The optimization of levodopa pharmacokinetics and avoidance of interaction with proteins; improvement in gastrointestinal dysfunction such as dysphagia and constipation; prevention and treatment of nutritional deficiencies either the micronutrients or vitamins could systematically be employed [55]. However, other therapeutic interventions such as continuous pump therapies with apomorphine or parenteral levodopa or the implantation of electrodes for deep brain stimulation could also be considered [8]. **Table 3** shows the type of protein detected in some popular neurodegenerative diseases, including those not discussed in this work.

*Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

#### **3.3 Huntington disease**

The disease got its name from the physician George Huntington, who first described it in late 1872. Huntington's disease is a hereditary, autosomal dominant, progressive neurodegenerative disease associated with a single abnormal gene on chromosome 4. The pathogenesis is initiated by a CAG (glutamine) trinucleotide expansion in exon 1 of the Huntingtin (*HTT*) gene, which is found at the short arm of chromosome 4p16.9. The normal function of the Huntington gene *HTT* is not known, but it may be involved in sustaining the cyclic adenosine monophosphate response element-binding protein, intracellular signaling, and obviating toxicity of neurons. Earlier studies suggest that the conjugation of the striatum-protein-rich Ras homolog with mutant HTT (mHTT) could lead to cellular toxicity. Although, why this protein causes cellular toxicity is poorly understood. Some evidence suggests that the interaction of the mHTT protein and the group 1 metabotropic glutamate receptors may be at the root of the delayed onset [11, 12, 56].

Huntington's disease (HD) is clinically characterized by cognitive dysfunction, abnormal involuntary movements, behavioral disturbance, and psychiatric disease. In abnormal involuntary movements, symptoms may include chorea, dystonia, rigidity, akathisia, bruxism, swallowing disorders, myoclonus, impaired manual dexterity, impaired global motor capacities, and gait and balance disorders. Cognitive dysfunction may present with impaired executive functions, bradyphrenia, language and communication disorders, and social cognition impairments. Behavioral disorders could be associated with memory disorders, disorientation, and visuospatial and visual perceptual disorders. Psychoanalysis of the patient could reveal depression, suicidal ideation or attempts, irritability, apathy, anxiety, obsessions, impulsivity, sexual disorders, hallucinations, sleep disorders, urinary incontinence, pain, dental pain, excessive perspiration, weight loss, hypersalivation, reduced lung function, and respiratory muscle strength [11, 12, 57].

The disease typically lasts 15–20 years, with dementia, mutism, dystonia, and bradykinesia becoming the classic symptoms in advanced forms of the disease. The mean age at onset is between the third and fifth decade of life, with a range of 2–85 years. Juvenile Huntington's disease (JHD) is when the first symptoms and signs appear before the second decade of life. The symptoms of young and old patients vary, as the younger patient presents with an overwhelming rigidity (Westphal variant), while the geriatric becomes bed-bound with rigidity and flexion contractures in the limbs [11, 58].

Pathologically, diffuse neurodegeneration is seen in the cortex and the striatum. The medium spiny neurons are the primary neurons affected, marked with the conspicuous presence of γ-aminobutyric acid and enkephalin. These neurons typically project into the lateral globus pallidus. With time, this degenerative process progresses to the rest of the basal ganglia with subsequent dissemination, reaching the cortex and substantia nigra. Aggregates of mHTT are seen within the nucleus and cytoplasm during microscopy. Inclusion bodies containing a complex of Huntingtin and other soluble mHTT are seen in the neurons. The cause of the cell death seen in this disease is yet to be delineated between the accumulation of the mHTT conglomerate or the soluble form of the protein when toxic. Glutamate, dopamine, and γ-aminobutyric acid are considered the most affected neurotransmitters in HD; hence, they are the focus of current pharmacological interventions [11, 12, 58].

The prevalence among the European is at 4–8 cases per 100,000, While America has not had a general epidemiological study since 1993. It is rare in Japan and Finland but common in Scotland and Venezuela. At the same time, there are inadequate data from Africans, Black Americans, and those in Eastern Asia [58].

#### *3.3.1 Diagnosis*

After accessing the clinical signs and symptoms presented, a genetic test called predictive test is requested. A DNA test showing abnormal CAG expansion (or repeats) in the *HTT* gene can be used to confirm the diagnosis in symptomatic individuals. The CAG (cytosine (C), adenine (A), and guanine (G)) repeats seen in the juvenile HD is over 55 in most cases. While for the elderly, it is about 36–40. The longer the repeats, the younger the age of the patients [11]. Biomarkers could also be exploited. With biomarkers, identification of mHTT in CSF could be a positive indication [12].

#### *3.3.2 Management*

Due to the myriad of disorders involved in this disease, it benefits more from symptomatic management rather than a definitive cure. This symptomatic management which is multi-disciplinary in approach, includes physical therapy, gastrostomy device, and medications such as antidepressants and antipsychotics—the chorea benefits from atypical antipsychotic drugs, which include olanzapine and tetrabenazine. Irritability benefits from an atypical antipsychotic drug in severe cases, but in mild cases, the use of selective serotonin reuptake inhibitor (SSRI), an antidepressant, may suffice. For obsessive–compulsive thoughts and actions, experts recommend SSRIs. For dystonia, physical therapy and injection of botulinum toxin are advocated [11, 58].

Since this disease involves the production of aberrant proteins, targeting the DNA or RNA may form a basis for drug discovery [12].

#### **4. Metabolic disorders**

Most metabolic disorders are nutrition-based disorders that are a result of the diet and lifestyle of the patients. Among these nutrition-based diseases, some ailment result from undernutrition due to food scarcity, leading to insufficient energy. At the same time, some result from overnutrition due to the insufficient capacity of the hormones regulating these nutrients. In this section, the focus is made on protein-energy malnutrition and diabetes mellitus, a type of hormonopathy.

#### **4.1 Protein-energy malnutrition**

The World Health Organization considers malnutrition in the context of both undernutrition and overnutrition. It could be described as the cellular imbalance between the supply of nutrients and energy and the body's demand to ensure healthy development, maintenance, and specific functions [59]. The term protein-energy malnutrition (PEM) includes kwashiorkor, marasmus, and intermediate states of marasmic-kwashiorkor. Those below 5 years may present a mixed picture of marasmus and kwashiorkor or milder forms of malnutrition. Malnutrition among children leads to waned immunity and increases susceptibility to diseases. Inadequate access to nutritious foods due to rising food prices is a common cause of malnutrition [60]. In the former times, rising food prices or food scarcity was induced by war; however, in recent times, terrorism and climate change could be implicated. Areas close to the desert may be experiencing an acute food shortage due to severe drought and other effects of climate change.

#### *Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

Studies have shown that those between 6 and 12 months are most affected, with over half of this population studied presenting with PEM and a third of those 13–24 months having PEM. Among these studies, marasmus is the most prevalent form of PEM, affecting a third of the population studied. Diarrhea and malaria are the associated co-morbidities popular with this disease, with over 60% of these populations coming from the lower socioeconomic status. The case fatality rate was 40.1%, with the males having more prevalence at 50.9%. Mortality among the marasmickwashiorkor and the unclassified group was 53.3 and 54.5%, respectively [61, 62].

The World Health Organization estimates that about two-thirds of all deaths occurring among pediatrics in developing countries could be attributed to malnutrition. Therefore, improving nutrition is crucial for reducing high infant and under-five mortality rates, the proportionate physical growth, the social and mental well-being of children, and academic achievement [61, 62]. Sub-Saharan Africa suffers the most from PEM around the world.

#### *4.1.1 Diagnosis*

The assay of total protein and albumin helps diagnose PEM, as early detection helps obviate the challenges associated with severe forms of PEM. In the final stage of wasting, reduced plasma albumin concentration ensues due to the adaptation of the human system to a protein-deficient diet. The development of marasmus reveals energy deficiencies in the diet, which leads to the change of the regular pattern of proteins. It is also observed that a decrease in serum albumin and total protein in PEM was due to reduced synthesis of protein resulting from inadequate intake of dietary protein. PEM in children is associated with a more significant deficiency of total protein, which may be as low as 50% of the child's total protein in severe cases. These reductions of total serum protein and albumin are prominent in kwashiorkor and marasmus [63].

In the absence of a diagnostic facility, the nutritional status of children is determined by clinical examination, history, and anthropometric measurements, which include height-for-age, weight-for-age, weight-for-height, head circumference, mid-upper arm circumference, and skinfold thickness which could be compared to the reference charts of the World Health Organization [60].

#### *4.1.2 Management*

A healthy balanced meal is advocated to fortify the immunity of infants and children under the age of 2. However, due to the inability to decipher the best description of a healthy balanced meal for children of such ages in most rural areas, exclusive breastfeeding is strongly advised for the first 6 months after delivery. While locally available meals and fruits rich in vitamin C, coupled with proteins such as eggs, are augmented with breastfeeding between the first 6 and 24 months of delivery. This daily intake of an egg and vitamin C-rich foods (or tablets) for at least one month is based on the need to boost the immunity and replenish the worn-out tissues of these pediatrics [64, 65]. The prevention of PEM cannot be overemphasized as it is associated with a high mortality rate among children under the ages of five in Sub Sahara, Africa [61, 62].

#### **4.2 Hormonopathy**

Hormonopathy is a term used in describing a disease associated with a change in the production of hormones. In these conditions, there can be over-secretion or under-secretion of hormones or even the production of aberrant proteins. Endocrine proteinopathies, which are grouped under hormonopathy, are diseases associated with peptide- or protein-derived hormones. They are characterized by the hyper- or hyposecretion of these proteins or an aberration in their structure and function. Proteinderived hormones include insulin, prolactin, ACTH, gastrin, parathyroid hormone, oxytocin, leptin, ADH and growth hormone. This section focuses on the most prevalent endocrine proteinopathy related to insulin, a disease called diabetes mellitus.

#### *4.2.1 Diabetes mellitus*

Diabetes causes severe life-threatening complications, such as hyperglycemic coma, hypoglycemic coma, severe impairment of renal function, blurred vision, memory loss, insulin allergy, and acute neuropathy. Managing it requires dietary control, physical exercise, and insulin administration. Demographic data is based on the patient's age, sex, location, and income. Clinical data is divided into physical signs and laboratory results. Physical signs are those obtained via physical examination of the patient, like BMI (body-mass index), pulse rate, and blood pressure, while the laboratory results are based on the blood sugar levels [66, 67].

An expert system determined by a set of rules used to make decisions is known as a rule-based expert system. Developing this expert system requires a knowledge of the engineering process in which the rules used by human experts are collated and translated into an appropriate form for computer processing [66, 67]. The rule-based expert system is utilized in this section.

#### *4.2.2 Diagnosis*

Laboratory results are associated with laboratory tests, like blood and urine tests [66–70]. Using the rule-based expert system, diagnosis is classified into:

	- Fasting plasma glucose ≥7.0 mmol/L
	- Random plasma glucose ≥11.0 mmol/L
	- Fasting plasma glucose 6.1–6.9 mmol/L
	- Random plasma glucose 7.0–11.0 mmol/L

#### *4.2.3 Management*

The management of diabetes can be classified into three categories.


*Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

	- The drugs for obese and lean patients.
	- The specific drugs for patients with challenges with their organs, such as renal diseases, lactic acidosis, and liver disease [69, 70].

#### **5. Conclusion**

Early and accurate diagnosis of protein-related diseases saves cost and prevents rapid deterioration of these disease conditions. It also prevents the waste of time and resources used in managing a misdiagnosed condition. The exposition in this chapter should be an eye-opener to the public and stakeholders in the public health domain to harp on the need for early diagnosis, treatment, or management of these diseases for improved health for all.

Health professionals and researchers are encouraged to give further research attention towards discovering a cure and treatment of these protein-related diseases. This can be done by utilizing the knowledge garnered from protein synthesis and the post-translational modification of proteins.

This work which is a summary of the prevalent neurodegenerative diseases and some metabolic disorders has taken the first step to elucidate on how proteins formed the basis of some modalities involved in the diagnosis and treatment of a few of these diseases; the onus lies on researchers in this field to consolidate on it and bring succor to the ailing population.

#### **Acknowledgements**

Special thanks to all those who diagnose and manage protein-related diseases in Nigeria; to those children of Northern Nigeria who became the first experience in PEM; to the geriatrics in Mgbidi, Oru west, whose health challenges continue to be an inspiration.

#### **Conflict of interest**

The authors declare no competing interests.

#### **Notes/thanks/other declarations**

Ohanube A.K. Goodluck conceptualized the work and wrote the section on neurodegenerative diseases and protein-energy malnutrition; Obeta M. Uchejeso wrote the section on Hormonopathy and Introduction; Ikeagwulonu Richard wrote the abstract and conclusion. All authors reviewed the work and approved the final manuscript.

### **Author details**

Ohanube A.K. Goodluck1 , Obeta M. Uchejeso2 \* and Ikeagwulonu R. Chinaza3

1 Department of Biomedical Sciences, The Hochschule Bonn-Rhein-Sieg, University of Applied Sciences, Bonn, Germany

2 Department of Medical Laboratory Management, Federal School of Medical Laboratory Science, Jos, Nigeria

3 Department of Medical Laboratory Services, Alex Ekwueme Federal University Teaching Hospital, Abakaliki, Nigeria

\*Address all correspondence to: uchejesoobeta@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Protein Detection in Clinical Diagnosis and Management of Prevalent Neurodegenerative… DOI: http://dx.doi.org/10.5772/intechopen.101051*

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#### **Chapter 3**

## Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy

*Shenbagamoorthy Sundarraj, Gopalan Rajagopal, Balaji Sundaramahalingam, Madasamy Sundar and Ramar Thangam*

#### **Abstract**

Emerging proteomic technologies offer new insight in the study of malignant tumor to identify protein biomarkers for early detection, stratification, prediction and monitoring of treatment, as well as to detect target molecules for therapy. The tumor protein biomarker is responsible for the regulation of the cell cycle to promote cell proliferation and resistance to cell death. Important technologies include ELISA, immunohistochemistry, flow cytometry, western blot, mass spectrometry, protein microarray, and microfluidics for the study of screening, protein profiling, identification, qualitative and quantitative analysis of differential expressed oncoproteins relative to cancer tissues, counterparts at different stages of the disease from preneoplasia to neoplasia. It can also provide a detailed description of identifying tissue-specific protein biomarkers and to analysis the modification of protein activity in cancer conditions. In this chapter, we discuss current and emerging protein assays for improving cancer diagnosis, including trends toward advances in assay miniaturization, improve sensitivity and specificity, time and cost-effective, and accuracy in detection and measurement of protein activity. However, information from these protein diagnostic technologies should be integrated to obtain the optimal information required for the clinical management of a patient.

**Keywords:** cancer, protein biomarker, ELISA, protein microarray, mass spectrometry

#### **1. Introduction**

Cancer is the leading cause of death global population. As stated by the National Cancer Institute annual report revealed that there were 18.1 million new cases and 9.5 million cancer-related deaths globally in 2018. By 2040, the number of new cancer cases per year is anticipated to arise around 29.5 million and the number of cancer-related deaths to 16.4 million [1]. Cancer mortality can be reduced if cases are detected earlier and treated systematically and can result in a greater probability of survival rate and less morbidity [2]. Cancer diagnosis and prognosis have advanced

dramatically during the last decades. Achieving this goal will necessitate not only improved therapies, but also enhanced methods for evaluating an individual's risk of developing cancer, detecting cancers at an early stage when they can be treated more effectively, distinguishing aggressive from non-aggressive cancers, and monitoring recurrence and response to therapy.

Diagnostic imaging technologies can be used to detect people with cancer, these tests can be physically invasive, time-consuming and expensive to screen large groups of people who are asymptomatic and can cause unnecessary stress and worry. Furthermore, diagnostic imaging technologies frequently overlook minor lesions, resulting in the disease not being detected until it has progressed to the point when treatment intervention is less effective. However, insufficient diagnostics prohibit the detection of certain types of cancer until the advanced stage. For instance endoscopy with biopsy is the distinctive screening method for esophageal cancer and is generally performed after symptoms appear [3]. Other screens may be providing high levels of false positives or negatives. Hepatocellular carcinoma is generally detected by ultrasound, but this technique is subjected to operator mistake and often cannot distinguish between malignant and benign nodules [4]. Although mammography is the standard screening techniques for breast cancer, 20% of cases go undetected with this screening and specificity is 25%, leading to a large number of false positives [5].

Improving strategies for screening asymptomatic individuals for early-stage malignancies is a particularly difficult challenge. Overcome these challenges in the recent years, there has been a surge in interest in molecular markers as a cancer diagnosis, prognosis and therapeutic response [6]. The cancer antigen 15–3 (CA 15–3) act as a potential biomarker is used to screening and monitoring breast cancer [7]. The prostate-specific antigen (PSA) is a widely mentioned marker that is used to test male patients for prostate cancer [8]. The analysis of overexpression of human epidermal growth factor receptor type 2 (Her2) and estrogen receptor levels in breast cancer patients [9, 10].

Specifically, some modern molecule-oriented techniques used protein as a biomarker for monitoring of cancer progression and early tumor detection. Furthermore, tumor biomarker protein assays are suitable method for holding important clinical diagnostic tests in future because gene level studies may not correlation for the cancer alteration [11, 12]. Protein biomarkers played significant roles in accurate early diagnosis, therapy and prognosis in colorectal cancer [13].

Serum protein biomarkers are well developed tools for cancer diagnosis [14]. As proof, prostate-specific antigen (PSA), cancer antigen 125 (CA-125) and carcinoembryonic antigen (CEA) is extensively used for the diagnosis and management of various types of cancer, namely prostate, ovarian and gastrointestinal cancers [15]. The clinical sensitivity of a biomarker can simply be defined as the proportion of people with a confirmed disease who test positive for the biomarker assay whereas specificity refers to the proportion of healthy individuals who test negative for the biomarker assay [16]. Noninvasive assays, such as those using blood, stool, urine, or saliva are preferred because they cause less pain to the patient, have higher abidance rates and may be taken frequently for monitoring the treatment response [17]. Measurement of serum proteins ensures distinguishes between various types of malignancies from benign and thus leading imaging analysis, endoscopic examination and other diagnostic procedures and monitoring of the efficacy of the treatment.

The main objective of this chapter is to provide a new insight of emerging technologies based on protein detection for cancer diagnostic and prognostic. Proteins analytical techniques are especially suitable for the diagnosis of cancer are described here briefly along with their recent development. We also provide a brief description of techniques currently used to identify the protein activity in post-translational modification have been linked to cancer diagnosis and cancer progression.

#### **2. Methods of proteins detection in cancer**

The developments of proteomic patterns have emerged as effective method for diagnostics in the field of cancer proteomics in that it is represents new way for cancer detection and also clinically feasible. The enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) and flow cytometry system represent the most reliable, sensitive and widely available protein-based testing platform in the clinic for the diagnosis, prognosis and treatment monitoring of cancer [18, 19]. Other protein analyses techniques such as mass spectrometry, Protein array and Microfluidics are currently at the laboratory level setup extensively used for cancer research purpose but these techniques are being developed for clinical applications (**Figure 1** and **Table 1**).

#### **2.1 Enzyme-linked immunosorbent assay**

The enzyme-linked immunosorbent test (ELISA) has been widely utilised in regular clinical diagnostics and is still considered the gold standard for detecting

#### **Figure 1.**

*Schematic illustration of the different proteomic techniques for detection, identification, screening, protein profiling and modification of protein in various cancers.*



*Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

#### **Table 1.**

*List of cancer protein biomarkers and their detection methods in the current research practice.*

cancer protein biomarkers in physiological samples [60–62]. Despite advances in developing cost-effective and label-free novel ELISA-based methods for future use in point-of-care cancer diagnostics, prognostics and therapy monitoring offers promise for improving the early detection of breast cancer (**Figure 2**). In conventional ELISA

#### **Figure 2.**

*Flowchart of nanocomposite used as a biosensing substrate and detection in breast cancer using nanoparticle coated arrays and ELISA based electrochemical immunosensor. First, a drop of blood from breast cancer (BC) patients is subjected to an nanoparticle fabricated array for a high-throughput screening of cancer biomarkers in breast cancer. Second, promising cancer biomarker candidates are selected from the array screening and validated in a large cohort of patients using ELISA, which can be used for early diagnosis, disease stratification, prediction of disease progression, or monitoring of drug responses. Finally, according to the function of nanoparticle coated array biomarker panel, biosensors could be designed and fabricated for clinical use in breast cancer.*

techniques, colorimetric or fluorescent readout signals are utilised to show FDAapproved protein biomarkers currently used in clinical practice (**Table 2**).

Stevens et al. generated a novel ELISA-based technique called plasmonic ELISA that uses gold nanoparticles as the probe to detect PSA in prostate cancer diagnosis [63, 64]. Shim et al. described a microfluidic droplet-based extremely flexible and sensitive diagnostic device for counting individual analyse molecules and identifying a biomarker for prostate cancer in buffer with a detection limit of 46 fM [65]. Zhang et al. described the estimation of PSA in a sandwich-type electrochemical ELISA with fabrication of PtNP-ferrocenedicarboxylic acid based infinite coordination polymer (ICP) in combination with polyamidoamine dendrimers modified sensor electrode [66]. Xu et al. described the simultaneous detection of triple cancer biomarkers, namely PSA, CEA and AFP using newly developed carbon and gold (CGN) nanocomposite-based immunoprobes [67].

We mainly focus on the recent advances made by a various group in improvement strategies for electrochemical ELISA-based immunosensors for improving access to diagnostics, increased detection sensitivity and specificity, magnification of the signal, ease of handling, potential for automation and combination with miniaturized analytical systems, low cost and comparative simplicity for mass production. The development of generation and characterization of double nanobody-based sandwich ELISA for the detection of FGL1 in cancer patient serum [41]. San Martin et al., developed the "gold standard" ELISA-based electrochemical immunosensor for the


*Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*



*Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

#### **Table 2.**

*List of FDA-approved protein tumor markers currently used in clinical practice.*

single determination of both proteins PD-L1 and HIF-1α in terms of assay time, compatibility making their use suitable for untrained users at the point of attention [40]. The fabrication of sandwich ELISA type electrochemical aptasensor is developed for the instantaneous determination of two important biomarkers arcinoembryonic antigen (CEA) and cancer antigen 15–3 (CA 15–3) in breast cancer. CEA and CA 15–3 aptamers linked to gold nanoparticles/redox probe/graphene nanocomposite were used as biosensing probes for signal amplification and to enhance the sensitivity of the immunoassay [33]. Poly(ε-caprolactone) electrospun scaffolds (ePCL) are used to arrange for a microstructured substrate with a high surface-to-volume ratio, capable of binding E7 oncoproteins when used for enzyme-linked immunosorbent assay (ELISA) tests [31]. Interestingly, the ultrasensitive detection of cancer biomarker matrix metalloproteinase-1 in urine, saliva, bovine serum, and cell culture media of oral and brain cancers using label-free electrochemical immunosensor based on gold nanoparticle/ polyethyleneimine/reduced graphene oxide nanocomposites [29]. Li et al., 2021 constructed a simple label-free electrochemical immunosensor based on worm-like platinum with a sandwich-like structure [21]. The fabricated electrochemical immunosensor showed a wide linear range, enhanced detection limit, good selectivity and stability for the determination of alpha-fetoprotein.

Applications of Enzyme-linked Immunosorbent Assay in cancer research


#### **2.2 Immunohistochemistry**

Immunohistochemistry (IHC) is a fundamental method used for clinical decision making of diagnosis and prognosis of various cancers, such as breast [68], prostate [69], lung [70, 71]. It enables to find out the analysis of biomarker expression and tissue localization in cancer. Immunohistochemical techniques play critical roles a diagnosis and screening tools for distinguishing between malignant and benign with the help of biomarkers expression in lung cancer [72]. In recent years, the advancement of microfluidic-based immunohistochemistry represents clinically validated approaches to the standard chromogenic staining for rapid, accurate, and automated breast cancer diagnosis [73]. The automated chromogenic multiplexed immunohistochemistry assay approach provides an exclusive sample-sparing tool to characterize limited tissue samples in lung cancer and making it an emerging method in the clinical analysis for therapeutic decision making of advanced NSCLC, provided that validation in a larger population is performed. This implies limiting the number of tissue slides despite the existence of specific and sensitive biomarkers (ALK, ROS1, BRAF V600E, PD-L1) and the obligation to distinguish lung adenocarcinoma from squamous cell carcinoma [74].

Applications of Immunohistochemistry in cancer research

• To predict the prognosis of tumors by identification of enzymes, tumor-specific antigens, oncogenes, tumor suppressor genes, and tumor cell proliferation markers.


#### **2.3 Flow cytometry**

Flow cytometry is a versatile technique with applications in a variety of fields, including immunology, virology, molecular biology, cancer biology, and infectious disease surveillance. Flow cytometry is a technique for swiftly analysing single cells that are suspended in a buffered salt solution and flow through one or more lasers. Each cell is subjected to a visible light scattering analysis as well as one or more fluorescence parameters. Visible light scatter is assessed in two directions: forward (Forward Scatter or FSC), which shows the cell's relative size, and at 90° (Side Scatter or SSC), which reveals the cell's internal complexity or granularity. Fluorescence measurements are performed on samples that have been transfected and expressed with fluorescent proteins (for example, Green Fluorescent Protein, GFP), stained with fluorescent dyes (for example, Propidium Iodide, DNA), or stained with fluorescently attached antibodies (e.g., CD3 FITC). It enables simultaneous characterization of mixed populations of cells from blood and bone marrow as well as dissociable solid tissues such as lymph nodes, spleen, mucosal tissues, and solid malignancies.

The availability of new reagents has resulted in an explosion in the number of parameters utilised in flow cytometry investigations during the last several years. The number of fluorochromes used to conjugate monoclonal antibodies has increased dramatically, including tandem dyes and polymer dyes. Additionally, the number of fluorescent proteins accessible for transfection beyond GFP has increased, including mCherry, mBanana, mOrange, and mNeptune. These advancements in fluorochromes and technology have enabled tests with over 30 parameters to be performed. Data analysis is the final step of a flow cytometry experiment. The two-parameter histogram (dot plot) gating and analysis method is still widely utilised. However, as the number of factors and complexity of experiments expand, newer cluster data analysis techniques such as PCA, SPADE, and tSNE are being used. These enhanced data mining techniques enable the extraction of relevant information from the highdimensional data generated by flow cytometry.

Since Mack Fulwyler initially invented the present kind of flow cytometers in 1965 [75], flow cytometry has now been used in quite a broad range of clinical areas for assessing protein expression in cancer cells [76, 77]. It is commonly used to diagnose of acute lymphoblastic leukemia [78]. Flow cytometry is a rapid and sensitive diagnostic method that makes it possible to characterize more satisfactorily the heterogeneous group of acute lymphoblastic leukemias. Flow cytometry has been historically used to detect the expression of CD56 in the diagnosis of chronic myelomonocytic leukemia (CMML). CD56 is a cell surface marker that presents the surface of monocytes [79]. Following over a decade of extensive research, high-throughput

image-based flow cytometry is now an accepted and widely used tool in scientific research, particularly in the field of cancer biology. Many researchers have replaced microscopy-based clinical tools with image-based flow cytometry. Erber's team originally used image-based flow cytometry to identify the presence of promyelocytic leukemia (PML) bodies for the diagnosis and prognosis of acute myeloid leukemia (AML) [80].

Applications of Flow cytometry in cancer research


#### **2.4 Western blot**

The Western blot (WB), also known as immunoblot, is an analytical and quantitative method for detecting particular proteins in various biological materials, including liquids and tissue/cellular homogenates [81]. Harry Towbin and colleagues developed the WB method in 1979. The WB approach provides clear and valuable information for assessing the phosphorylation state of a protein. We can evaluate the modified form of protein in the sample either qualitatively or quantitatively. Radenkovic et al., detected cyclin D1 expression in tumour and peritumoral tissue of breast cancer patients by Western blotting method to found that Cyclin D1 expression decreased significantly with each advanced clinical stage of disease and tumour size [25]. Kinase activity-tagged western blotting (KAT-WB) detected autophosphorylation of Tyr-kinase and site-specific phosphorylation by multiple kinases enables to interrogate multiple kinase signaling pathways without using radioactive substances [82]. Western blot analysis provides an opportunity to obtain more insight into cell cycle regulation factor in tumorigenesis, could spur the discovery of many more successful therapeutic targets [83].

Applications of Western blot in cancer research


*Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

#### **2.5 Mass spectrometry**

The emphasis use of mass spectrometric analysis in clinical research has been on biomarker identification, which includes proteomics, lipidomics, and metabolomics [84, 85]. Metabolites, proteins and lipids have been shown to help distinguish between malignant and healthy tissue among the many compounds detectable with MS. To be identified as a qualifying biomarker, a molecule must be distinguishable from other molecules, ideally, the sample is simple, quick, and easy to collect, high sensitivity and specificity [86, 87], and is used to diagnose and prognosis for various cancer such as thyroid cancer [88], lung cancer [89, 90], bladder cancer [91], Pancreatic Cancer [92, 93], breast cancer [94], ovarian cancer [52], oral cancer [53], prostate cancer [95]. In general, various classes of molecules may function as biomarkers due to an imbalance of tumor-suppressing and promoting agents in cancer cells, regulating genetic changes, and changing the composition of lipids, metabolites, and proteins (**Figure 3**).

The MALDI-TOF MS combined with magnetic bead used for detecting serum protein biomarkers and establishment of boosting decision tree model for diagnosis of colorectal cancer patients [96], breast cancer [97]. These differentially regulated proteins were considered as potential biomarkers for the patients with CRC in the serum. The emerging mass spectrometry methods of nanoLC–MS/MS, targeted LC–MS/MS, and stable isotope-labeled multiple reactions monitoring (MRM) MS coupled to test

#### **Figure 3.**

*Schematic representation showed that well established method immunohistochemistry was widely used in clinical diagnosis for cancer detection. In recent years, emerging proteomic technologies such as mass spectrometry and protein microarray has been developed for precise detection of cancer protein biomarkers.*

machine learning algorithms and logistic regression used to analyze plasma samples from colorectal cancer patients. The novel peptide biomarkers combination of PF4, ITIH4, and APOE achieved sensitivity 84.5%, specificity 97.5% and an AUC of 0.96 in CRC diagnosis [98]. Moran et al., developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from prostate cancer patients' urine [99, 100].

The integrating mass spectrometry imaging and gold nanoparticle (AuNP)-based signal amplification was developed for quantitatively profiling protein biomarkers on the surface of exosomes in cancer diagnosis. Cancer protein biomarkers were modified with organic oligomers as mass tags and specific antibodies on AuNPs. Exosomes captured by the antibody-coated gold chip are recognized by the AuNPs probes, forming a sandwich immunoassay. Multiple protein biomarkers can be quantitatively detected from the exosomes with the mass tags by mass spectrometry imaging [101]. Park et al., 2019 developed a simple and robust cancer diagnostic method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based total serum protein fingerprinting to diagnose liver cancer [102]. The proteome profile of vimentin, tubulin beta 2C chain, tubulin alpha 1C chain, actin cytoplasmic 2, apolipoprotein A-I, and collagen alpha 2(VI) chain as a potential biomarker that exhibited differential expression in ovarian cancer using two-dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ ionization-time of flight mass spectrometric (MALDI-TOF MS) analysis [103]. The protein modifications in the N-glycosylation profile are usually associated with many cancers, like colorectal cancer. In turn, MALDI-TOF/MS and LC–MS methods are the most accurate technology in the quantification of N-glycans compositions in the serum and tissue of colorectal cancer patients [104]. The similar proteomic analysis was performed to identify potential lung cancer biomarkers such as CD5L, CLEC3B, ITIH4, SERFINF1, SAA4, SERFINC1, and C20ORF3 detected via a liquid biopsy-for the noninvasive diagnosis of lung cancer [105].

Applications of mass spectrometry in cancer research


#### **2.6 Protein array**

The analysis of protein biomarkers using these high throughput methods can provide robust and previously unachievable diagnostic and prognostic information

#### *Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

for a variety of cancers [106, 107]. The analytical protein array is a useful developing technology that enables simultaneously analyzing >4000 protein samples in cell or tissues for biomarker discovery (Huang et al., 2017). The microarray is currently utilized to analyze biopsy samples in clinical research trials, essentially lead to the collection of information linked to posttranslational modifications of proteins reflecting the active status of signal pathways and networks [108]. This technique has the potential to enhance cancer detection, prognosis, and treatments. Protein microarray technology has been used effectively in fundamental and applied proteome research and affinity studies for protein identification, quantification, and functional analysis [109, 110]. A protein function array is made up of thousands of natural proteins that have been immobilised in a specific arrangement. When a functional protein array is used for serum protein profiling, autoantibodies are usually detected as biomarkers for diagnosis of cancer detection and for monitoring the cancer treatment due to their stability, specificity, and ease of detection, as compared with other serological components [111]. Protein microarrays have allowed researchers to examine functional protein dysregulation in various cancer namely colorectal cancers [112], pancreatic cancer [113]. Mirus et al., identified ERBB2, TNC and ESR1 in prediagnostic plasma from people that succumb to pancreatic ductal adenocarcinoma [114]. In addition to the understanding of the biological mechanisms, analytical protein arrays have also been applied to profile drug resistance [115].

In recent advancements in protein microarray is Reverse-phase protein array/ microarray (RPPA/RPPM), which can precisely map functional proteomic profiling in individual cancer patient. The personalized therapy was prescribed by the identification of functional proteomics profilling. RPPA is an antibody-based highly quantitative proteomic technology, used for profiling the expression and modification of signaling proteins, mainly in low-abundance analytes cases. Clinical trials are using RPPA technology molecular-targeted therapeutics [116, 117]. Horton et al., 2021 found that the minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables have been successfully managed in a multi-site clinical trial setting for leukemia [118]. A proteomic study was carried out for determining the levels of post-translational protein modifications and total protein expression in myeloproliferative neoplasms patients using RPPA [119]. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of cancer or response to therapy. Recent proteomics studies have focused on the expression of seven markers (CD5, CD10, BCL2, BCL6, MUM1, Ki-67, and C-MYC) is analyzed by RPPA using 37 diffuse large B-cell lymphomas (DLBCL) tissues [120]. These results suggest that RPPA could be applicable as a supportive tool for determining lymphoma prognosis. With all of these improvements, we believe that protein array technology will soon become a dominant tool for biomarker discovery in cancers.

Applications of protein array in cancer research


#### **2.7 Microfluidics**

Microfluidic technology, as new creativity has a great impact on automation and miniaturization via handling a small volume of materials and samples for cancer diagnosis [121]. This method has be considered as a primary screening tool for diagnosing breast cancer based on its robustness, high throughput, low energy requirements, excellent accuracy and accessibility to the general public [122]. A miniaturized instrument was developed for chemiluminescence detection and signal analysis with the advances in microfluidic technology. The system was validated by testing four biomarkers of colorectal cancer using plasma samples from patients [123]. Another design of the microfluidic device, magnetic nanoparticles (Fe3O4NPs) was successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. The captured exosomes were then subjected to analysis of CA19–9, a protein often used to monitor pancreatic cancer patients [124]. In the line of discovery, the nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples was detected using the microfluidic paper-based analytical device (mu PAD) was developed by Jiang [125]. This method is feasible for home-based self-detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response (**Figure 4**). The functions and recent development of microfluidic chip to provide great potential for advancing noninvasive cancer diagnosis [126, 127].

#### **Figure 4.**

*Microfluidic technology, as new creativity has a great impact on automation and miniaturization via handling a small volume of materials and samples for cancer diagnosis. An effective management of cancer diagnosis screening by using body fluids and cancer protein biomarkers for diagnosis, prognosis, therapy and monitoring to treatment.*

#### *Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

Microfluidic devices which used to mimic cancer metastasis process are usually applied to several cell types in order to culture two or more organoids. Different organoids are separated by some specific biomaterials, such as polydimethylsiloxane (PDMS), and connected with each other by channels and controllable fluids. Xu et al. designed and constructed a multi-organ microfluidic chip to mimic lung cancer metastasis to the brain, bone and liver. In this platform, organoids were divided into different chambers, including upstream lung organoid and three downstream organoids. Different types of cells were seeded in each chamber to culture different organoids and each organoid were linked by side channels. The culture medium flowed through microvascular channels to simulate blood circulation. At the same time, a circulating vacuum was applied to mimic the physiological breathing [128]. This system provided a physiologically relevant context to recapitulate the complex process of lung cancer metastasis and help us to effectively explore the underlying mechanism of lung cancer metastasis.

The integration between 3D bioprinting and microfluidic chip has given microfluidic chip greater potential to model cancers. Traditionally, in cancer modeling on chip, microfabrication such as micromachining, photolithography and injection molding, are used in the fabrication of microfluidic chips [129]. These methods have high resolution and accuracy, but their high cost, complex process and difficult reproducibility greatly limited the development of microfluidic chip [130]. The emerging of 3D printing technology greatly simplifies the fabrication process of microfluidic chips. The biggest characteristic of microfluidic chip is the customizability, which means microfluidic chip is a very flexible scientific tool that can accommodate with advanced technologies. To date, microfluidic chip shows tremendous promise in cancer diagnosis and treatment. Microfluidic chip can be applied in everything from anticancer drug development and screening to cancer modeling and diagnosis.

Applications of Microfluidics in cancer research


#### **3. Conclusions**

Over the past decade, protein profiling has emerged as a dynamic discipline, capable of generating a comprehensive perspective of protein patterns, modification of protein in various tissue-specific cancer types and mechanisms of cancer progression. Proteomics studies and analytical techniques are developed with modern materials for precise detection of tumor-specific alteration in proteins. Recent times, the proteomics technologies have fabricated with modern nanocomposites provide a new and more efficient methods for protein detection and identifying biomarkers for the early detection of cancer. The upgraded proteomics technology will modify the current pathological classification and grading methods of cancer during the next decade. Proteomic technologies will have an impact on the diagnosis and management of cancer.

### **Author details**

Shenbagamoorthy Sundarraj1 \*, Gopalan Rajagopal1 , Balaji Sundaramahalingam1 , Madasamy Sundar2 and Ramar Thangam3

1 Postgraduate and Research Department of Zoology, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

2 Centre for Research and Postgraduate Studies in Botany, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

3 Department of Materials Science and Engineering, Korea University, Seoul, Republic of Korea

\*Address all correspondence to: sundarrajbu09@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Methods of Protein Detection in Cancer for Diagnosis, Prognosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.101050*

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Section 2
