**2. Molecular approach**

**Figure 2.** Schematic representation of the classical strategy employed in initial studies of the *Lonomia obliqua* venom. The bristle extract was analyzed through denaturing electrophoresis (SDS-PAGE) which showed the venom is a com‐ plex mixture of proteins. Screening assays were carried out to investigate possible effects on blood coagulation and fibrinolysis. The venom showed procoagulant activity by decreasing blood clotting time. Two procoagulant compo‐ nents (Lopap, a prothrombin activator and Losac, a factor X activator) were identified and isolated from the bristle extract for further characterization. The specific activity of Lopap and Losac were observed on purified coagulation factor zymogens (FII or FX) using chromogenic substrates to detect generation of active forms of clotting factors (FIIa and FXa) by these toxins. This assay was used in the purification process to identify the active fractions containing each toxin. SDS-PAGE profile shows Lopap (1- multimer, 3- monomer) and Losac (2) are abundant components in the ven‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Production of recombinant forms of *Lonomia* toxins and discovery of new molecules are opening perspectives in the scientific area for basic and applied researches. These molecules can point out novel mechanisms of action, undiscovered molecular interactions and new classes of enzymes and inhibitors. Interesting, some venom toxins have shown multifunctional properties [19, 22, 28]. The best examples are Lopap (a prothrombin activator with high similarity with lipocalins) and Losac (a factor X activator highly similar to hemolins). Besides activation of blood coagulation, Lopap and Losac can modulate cellular functions and promote cell survival [22, 45]. Both molecules were cloned and produced in its recombinant form in

Additional studies will be conducted to determine the involvement of the venom components in the envenoming syndrome and their biological significance for physiological processes of the animal, such as insect metamorphosis, which is a combination of growth/activation/ differentiation/programmed cell death signals. Thus, this chapter reviews the currently

om. MM: molecular markers.

Applications

178

yeast and/or bacteria [19, 25, 28].

For many years, direct purification of toxins from venoms was the best procedure to charac‐ terize them with regard to their primary structure. Then, the development of molecular approaches to characterize toxin genes represented an expansion in the understanding of the structure and function of toxin, critical for the development of new treatments directed against the venom toxins (antivenoms). Cloning of cDNAs coding for biochemically isolated toxins has improved their characterization. *Transcriptomic* allows the identification of cellular transcripts in a given cell population, while proteomic studies protein's properties and functions (expression level, structure, post-translational modification, etc.) of proteins expressed by the genome of an organism at a certain point of time. The availability of tech‐ nologies for high throughput analysis has led to integrate toxin expression at mRNA and

protein level. This flow of genetic information from DNA to proteins is the base of the central dogma of molecular biology [46].

### **2.1. Transcriptomics of** *Lonomia obliqua* **bristle extract**

Expressed Sequence Tags strategy is an approach to characterize the transcriptome of a cell, gland or organism and is based in all the transcript (the most abundant are the mRNAs) produced at a specific time and fully sequenced to create a representative catalogue of expressed genes [47]. Hundreds to a thousand of sequences are grouped into *contigs* or clusters based on DNA sequence information and bioinformatics analysis (Figure 4). Nowadays, the EST-based strategy is commonly employed for identifying expressed genes in species of interest [48, 49]. This approach has been successfully used to compile a lists of genes expressed in venom's glands of a wide range of animals [50-53].

EST-strategy was used to identify the major transcripts present in *L. obliqua* bristle extract [19, 20, 43]. About 702 clusters (representing 1,278 independent clones) were assembled and characterized as lipocalins, hemolins, serine proteases, serine protease inhibitors, serpins, tumor suppressors, ribosomal, structural and cell cycle proteins as shown in Table 2 [20]. Most of the transcripts represent proteins involved in the animal physiology. Those sequences were deposited in data bank (NCBI GenBank accession numbers: CX815710–CX817210, CX820335– CX820336, AY908986) [20]. A pool of DNA sequences showed no similarities with well-known sequences in data bank. The most abundant toxin was a lipocalin of 21 kDa, and analysis of its N-terminal sequence shows 100% homology with Lopap (GenPept accession number: AAW88441). The Lopap whole sequence (accounting for 1.6% of the total clones) was identified in this cDNA library (accession number: AY908986).


Other cDNA libraries were constructed from bristles and integument [43]. The transcripts of those libraries revealed the presence of sequences related to trypsin-like enzymes, blood coagulation factors, prophenoloxidase cascade activators, cysteine proteases, phospholipase A2, serpins, cystatins, antibacterial proteins, lipocalins, and others (GenBank accession number: AY829732–AY829859) [43]. Sequences deposited independently in gene banks from both cDNA libraries are complementary. Apart from new venom component precursors, both

**Figure 4.** Schematic view of a transcriptomic approach based on EST-strategy. The strategy of construction of the cDNA library starts by the extraction of total RNA from a sample (ex. *L. obliqua* bristle extract). After purification of the mRNAs with an oligo (dT)-cellulose column, the cDNAs are synthesized (reverse transcription) by using synthetic oligo‐ nucleotides containing a restriction sites (in figure: NcoI and EcoRI for sense and antisense primers, respectively). The cDNA obtained can be inserted into a vector (plasmids or phages), generating a cDNA library. The library can be per‐ petuated by transforming the clones (plasmids) or infecting themselves (in the cases of phages) in *E. coli*. Based on DNA sequence information, bioinformatics tools predict the amino acid sequence of the corresponding gene products and their similarity to known genes. The redundant EST data sets are organized and integrated into cluster [54, 55].

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**Table 2.** Major transcripts present in the *Lonomia obliqua* bristle extract identified by EST-based strategy. Adapted from EST data-bank of NCBI deposited by Reis and colleagues in 2004 [19, 20].

protein level. This flow of genetic information from DNA to proteins is the base of the central

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Expressed Sequence Tags strategy is an approach to characterize the transcriptome of a cell, gland or organism and is based in all the transcript (the most abundant are the mRNAs) produced at a specific time and fully sequenced to create a representative catalogue of expressed genes [47]. Hundreds to a thousand of sequences are grouped into *contigs* or clusters based on DNA sequence information and bioinformatics analysis (Figure 4). Nowadays, the EST-based strategy is commonly employed for identifying expressed genes in species of interest [48, 49]. This approach has been successfully used to compile a lists of genes expressed

EST-strategy was used to identify the major transcripts present in *L. obliqua* bristle extract [19, 20, 43]. About 702 clusters (representing 1,278 independent clones) were assembled and characterized as lipocalins, hemolins, serine proteases, serine protease inhibitors, serpins, tumor suppressors, ribosomal, structural and cell cycle proteins as shown in Table 2 [20]. Most of the transcripts represent proteins involved in the animal physiology. Those sequences were deposited in data bank (NCBI GenBank accession numbers: CX815710–CX817210, CX820335– CX820336, AY908986) [20]. A pool of DNA sequences showed no similarities with well-known sequences in data bank. The most abundant toxin was a lipocalin of 21 kDa, and analysis of its N-terminal sequence shows 100% homology with Lopap (GenPept accession number: AAW88441). The Lopap whole sequence (accounting for 1.6% of the total clones) was identified

> **No. of clusters**

General Metabolism 72 94 1.30 6.1 7.0 Transcriptional and translational 165 462 2.80 30.7 37.0 Processing and sorting 10 13 1.30 0.8 1.0 Degradation 9 20 2.22 1.3 2.0 Structural functions 47 243 5.17 16.2 19.0 Cell regulation 26 82 3.15 5.4 6.0 Other functions 138 244 1.77 16.2 19.0 Conserved unknown proteins 42 120 2.86 8.0 9.0 TOTAL 509 1278 2.51 85.0 100.0

**Table 2.** Major transcripts present in the *Lonomia obliqua* bristle extract identified by EST-based strategy. Adapted

from EST data-bank of NCBI deposited by Reis and colleagues in 2004 [19, 20].

**No. of clones**

**Clones/ clusters**

**% of Total % of Hits**

dogma of molecular biology [46].

Applications

180

**2.1. Transcriptomics of** *Lonomia obliqua* **bristle extract**

in venom's glands of a wide range of animals [50-53].

in this cDNA library (accession number: AY908986).

**Functional categories**

**Figure 4.** Schematic view of a transcriptomic approach based on EST-strategy. The strategy of construction of the cDNA library starts by the extraction of total RNA from a sample (ex. *L. obliqua* bristle extract). After purification of the mRNAs with an oligo (dT)-cellulose column, the cDNAs are synthesized (reverse transcription) by using synthetic oligo‐ nucleotides containing a restriction sites (in figure: NcoI and EcoRI for sense and antisense primers, respectively). The cDNA obtained can be inserted into a vector (plasmids or phages), generating a cDNA library. The library can be per‐ petuated by transforming the clones (plasmids) or infecting themselves (in the cases of phages) in *E. coli*. Based on DNA sequence information, bioinformatics tools predict the amino acid sequence of the corresponding gene products and their similarity to known genes. The redundant EST data sets are organized and integrated into cluster [54, 55].

Other cDNA libraries were constructed from bristles and integument [43]. The transcripts of those libraries revealed the presence of sequences related to trypsin-like enzymes, blood coagulation factors, prophenoloxidase cascade activators, cysteine proteases, phospholipase A2, serpins, cystatins, antibacterial proteins, lipocalins, and others (GenBank accession number: AY829732–AY829859) [43]. Sequences deposited independently in gene banks from both cDNA libraries are complementary. Apart from new venom component precursors, both

libraries describe gene products related to cellular processes important for venom production, including high protein synthesis, tuned post-translational processing and trafficking. Those important projects contributed significantly to the characterization of this venom, which showed to be a rich source of proteins and active principles. Further studies about the biological and pharmacological properties of these molecules are necessary to understand its involve‐ ment in the envenoming process. Recently, the next-generation of sequencing methods - for example, pyrosequencing - have improved and increased the sequencing reducing time and cost compared to the traditional Sanger method [47, 56].

charge by addition of SDS detergent. This step also includes denaturation of proteins by reduction and alkylation. The second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), where smaller proteins migrate faster through the gel to the anode than larger ones.

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Immunoblotting involves the transfer of proteins from gel to a nitrocellulose or PVDF membrane in an electric field [62]. The immobilized proteins in the membrane are subsequently incubated with antibodies that have affinity for the proteins of interest. Detection is carried out by enzyme-labelled secondary antibodies against the constant region of the primary IgG antibody, followed by the addition of a chemilluminescent substrate. The substrate reaction

**Figure 5.** General approaches in Immunoproteomics. **Gel-based approach**: Bidimensional electrophoresis is based on protein separation on their pI and molecular mass. Then, the proteins are transferred from gel and immobilized on membrane (Western blotting). Antigens will be detected after serum incubation, followed by addition of secondary labelled antibodies and their substrates. **Gel-free approaches**: Antigen array: Proteins are fractionated (pI, hydropho‐ bicity,etc) and spotted in a solid support. After that, antigenic fractions can be detected using patient serum and sec‐ ondary labelled antibodies. Antigen profiling: Immunocapture is based on immobilization of patient immunoglobulins G, which are directly used to capture and isolate antigenic proteins from a complex mixture of pro‐

Following this, the immunogenic proteins are removed from the gel and enzymatically digested for further mass spectrometry analyses [63]. Trypsin is generally used, cleaving an amide bond on the C-terminal side of lysine and arginine residues, which will be protonated and analyzed in positive-ion mode. Addition of diluted acid (0.1% formic acid or 1% trifluor‐

The ionization methods that are most often used for peptides and proteins are Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI). Peptides and proteins

teins. Captured antigens are profiled by mass spectrometry (modified from 66).

acetic acid) to the sample contributes to the ionization process.

Detection of proteins can be performed by gel staining or immunoblotting.

can be visualized by fluorescence.

### **2.2. Microarray analysis**

The identification of genes expressed in cells of a tissue is a basic step to provide essential information about gene function and tissue physiology. The gene expression analysis through the microarray technology (cDNA arrays) has become a powerful tool for rapid analysis of the functional effects of toxins on cells and tissues [57]. The main application of cDNA arrays is to compare the expression of known genes in different physiological situations, for example, tissues in normal and pathological conditions [58]. Thus, analyses of array data contribute to a better understanding of complex gene expression patterns related to physiology and metabolism, unveiling networks or pathways previously unknown.

A study of the effects of *L. obliqua* bristle extract on the gene expression profile of cultured human fibroblasts showed that many genes are up- and down-regulated, especially those related to the inflammatory processes such as IL-8, IL-6, CXCL1 and CCL2 [44]. Other changes in the expression pattern of some genes, such as prostaglandin-endoperoxide synthase 2, urokinase-type plasminogen activator receptor and tissue factor, were also observed, which could contribute to the pathological effects of lonomism. The authors suggest that the clinical manifestations may be a result of the direct action of *L. obliqua* venom on the host cells allied to an indirect effect caused by alteration in the gene expression pattern in host tissues.

### **2.3. Immunoproteome of** *Lonomia obliqua* **bristle extract**

The identification of antigens eliciting an immune response by applying proteomic technolo‐ gies can be defined as *Immunoproteomics*. Some usual immunoproteomics approaches are shown in Figure 5. Here, the perspective for its application regards the improvement of serum therapy by the selection of antigens for toxin-specific immunization of horses. Furthermore, some applications correlate the identification of antigens with certain diseases, such as infectious, autoimmune or cancer, providing diagnostic and monitoring informations. In this way, these methodologies are good choices in developing clinical applications and also to discover biomarkers. [59].

In classical gel-based strategy, the isolation and identification of proteins/antigens comprises a combination of bidimensional electrophoresis, immunoblotting and mass spectrometry. The aim of bidimensional electrophoresis is to isolate proteins based on their charge and mass [60, 61]. The first step is isoelectric focusing (IEF), where proteins migrate to reach their isoelectric point in an immobilized pH gradient gel under high voltage. All proteins are given negative charge by addition of SDS detergent. This step also includes denaturation of proteins by reduction and alkylation. The second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), where smaller proteins migrate faster through the gel to the anode than larger ones. Detection of proteins can be performed by gel staining or immunoblotting.

libraries describe gene products related to cellular processes important for venom production, including high protein synthesis, tuned post-translational processing and trafficking. Those important projects contributed significantly to the characterization of this venom, which showed to be a rich source of proteins and active principles. Further studies about the biological and pharmacological properties of these molecules are necessary to understand its involve‐ ment in the envenoming process. Recently, the next-generation of sequencing methods - for example, pyrosequencing - have improved and increased the sequencing reducing time and

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The identification of genes expressed in cells of a tissue is a basic step to provide essential information about gene function and tissue physiology. The gene expression analysis through the microarray technology (cDNA arrays) has become a powerful tool for rapid analysis of the functional effects of toxins on cells and tissues [57]. The main application of cDNA arrays is to compare the expression of known genes in different physiological situations, for example, tissues in normal and pathological conditions [58]. Thus, analyses of array data contribute to a better understanding of complex gene expression patterns related to physiology and

A study of the effects of *L. obliqua* bristle extract on the gene expression profile of cultured human fibroblasts showed that many genes are up- and down-regulated, especially those related to the inflammatory processes such as IL-8, IL-6, CXCL1 and CCL2 [44]. Other changes in the expression pattern of some genes, such as prostaglandin-endoperoxide synthase 2, urokinase-type plasminogen activator receptor and tissue factor, were also observed, which could contribute to the pathological effects of lonomism. The authors suggest that the clinical manifestations may be a result of the direct action of *L. obliqua* venom on the host cells allied to an indirect effect caused by alteration in the gene expression pattern in host tissues.

The identification of antigens eliciting an immune response by applying proteomic technolo‐ gies can be defined as *Immunoproteomics*. Some usual immunoproteomics approaches are shown in Figure 5. Here, the perspective for its application regards the improvement of serum therapy by the selection of antigens for toxin-specific immunization of horses. Furthermore, some applications correlate the identification of antigens with certain diseases, such as infectious, autoimmune or cancer, providing diagnostic and monitoring informations. In this way, these methodologies are good choices in developing clinical applications and also to

In classical gel-based strategy, the isolation and identification of proteins/antigens comprises a combination of bidimensional electrophoresis, immunoblotting and mass spectrometry. The aim of bidimensional electrophoresis is to isolate proteins based on their charge and mass [60, 61]. The first step is isoelectric focusing (IEF), where proteins migrate to reach their isoelectric point in an immobilized pH gradient gel under high voltage. All proteins are given negative

cost compared to the traditional Sanger method [47, 56].

metabolism, unveiling networks or pathways previously unknown.

**2.3. Immunoproteome of** *Lonomia obliqua* **bristle extract**

**2.2. Microarray analysis**

Applications

182

discover biomarkers. [59].

Immunoblotting involves the transfer of proteins from gel to a nitrocellulose or PVDF membrane in an electric field [62]. The immobilized proteins in the membrane are subsequently incubated with antibodies that have affinity for the proteins of interest. Detection is carried out by enzyme-labelled secondary antibodies against the constant region of the primary IgG antibody, followed by the addition of a chemilluminescent substrate. The substrate reaction can be visualized by fluorescence.

**Figure 5.** General approaches in Immunoproteomics. **Gel-based approach**: Bidimensional electrophoresis is based on protein separation on their pI and molecular mass. Then, the proteins are transferred from gel and immobilized on membrane (Western blotting). Antigens will be detected after serum incubation, followed by addition of secondary labelled antibodies and their substrates. **Gel-free approaches**: Antigen array: Proteins are fractionated (pI, hydropho‐ bicity,etc) and spotted in a solid support. After that, antigenic fractions can be detected using patient serum and sec‐ ondary labelled antibodies. Antigen profiling: Immunocapture is based on immobilization of patient immunoglobulins G, which are directly used to capture and isolate antigenic proteins from a complex mixture of pro‐ teins. Captured antigens are profiled by mass spectrometry (modified from 66).

Following this, the immunogenic proteins are removed from the gel and enzymatically digested for further mass spectrometry analyses [63]. Trypsin is generally used, cleaving an amide bond on the C-terminal side of lysine and arginine residues, which will be protonated and analyzed in positive-ion mode. Addition of diluted acid (0.1% formic acid or 1% trifluor‐ acetic acid) to the sample contributes to the ionization process.

The ionization methods that are most often used for peptides and proteins are Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI). Peptides and proteins can be identified by Peptide mass fingerprinting (PMF) or *de novo* sequencing [63]. PMF is based on their fragmentation pattern, considering that identical peptide maps have identical amino acid sequences. For *de novo* sequencing analyses, the precursor ion is selected for fragmentation and the product ions are evaluated by mass differences between successive peaks in the spectrum, which are related to the individual mass of their residues.

The identification of immunogenic compounds from *L. obliqua*'s bristle extract was performed on gel-based approach using the polyclonal horse anti-Lonomic hyperimmune serum and anti-Lopap specific rabbit serum produced by the Butantan Institute [9]. Bidimensional electro‐ phoresis of bristle extract revealed 157 silver stained spots, under non-reducing conditions (without DTT and iodoacetamide addition), providing an overview protein mapping (Figure 6A). However, 153 spots were immunodetected using anti-Lonomic serum (Figure 6B) and 30 spots detected using anti-Lopap serum (Figure 6C). Abundant proteins from 24 selected colloidal Coomassie Blue gel spots, corresponding to immunogenic proteins, were digested with trypsin and analysed by tandem mass spectrometry. The identification searches were carried out using the *L. obliqua* bristle EST databank. Lipocalins (spots 05, 09, 10, 14, 15, 16, 18, 24), cuticle protein (spots 05, 06, 07, 08, 11, 12, 13) and serpins (spot 21) were amongst the proteins identified (Figure 6A) [9]. Lipocalins can play a role in homeostasis and inflammation, as a defense mechanism in haematophagous arthropods. Lopap, characterized as a lipocalin protein member, and its all isoforms were highly represented as immunogenic proteins, revealed by the specific anti-Lopap serum (Figure 6C). The bristle' cDNA libraries also confirm the high abundance of lipocalins. As previously described [9, 19], these proteins have impor‐ tant role in envenoming. The cuticle proteins identified can be related to the inflammatory response caused by macerated spicule proteins. The serpin protein may also be involved in the defense mechanism.

Besides the biochemical and pharmacological tests, the quality control of serum and vaccine production can be monitored by proteomic technologies [64], such as chromatographic analyses, bidimensional electrophoresis and immunoblotting, once they are able to detect protein degradation and also confirm the presence of specific antibodies. However, immuno‐ therapy can be more effective if a better characterization of venom composition is performed, improving immunization procedures, increasing its specificity and reducing side effects. The new generation of high affinity antibodies against low abundant immunogenic toxins can be evaluated by an antivenomic approach [64, 65].

A novel approach is the investigation of post-translational modifications (PTM) that affect antigen recognition, given that many peptides presented to T cells by the major histocompa‐ bility complex are post-translationally modified [66]. Glycosylation and phosphorylation are important PTMs of proteins, playing crucial roles in several biological processes, including cell recognition and signalling pathway [67, 68]. Some potential targets for cancer therapy are based on glycosylated and phosphorylated epitopes discoveries [59].

Otherwise, phosphorylated proteins are usually enriched by immunoprecipitation (mainly for phosphotyrosine peptides) or by chromatographic procedures, such as Strong Cation eX‐ change (SCX), Hydrophilic Interaction Liquid Chromatography (HILIC), Immobilized Metal Affinity Chromatography (IMAC) or Metal Oxide Affinity Chromatography (MOAC). **Figure 6.** Bidimensional electrophoresis and immunoblotting from *Lonomia obliqua*'s bristle extract. (A): silver stained bidimensional gel (100 μg of protein applied) under non-reducing condition. Panels (B) and (C): PVDF im‐ munoblotted 2D gels incubated with anti-Lonomic horse serum diluted 1:500 (B) or with anti-Lopap rabbit serum

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diluted 1:250 (C) [9].

can be identified by Peptide mass fingerprinting (PMF) or *de novo* sequencing [63]. PMF is based on their fragmentation pattern, considering that identical peptide maps have identical amino acid sequences. For *de novo* sequencing analyses, the precursor ion is selected for fragmentation and the product ions are evaluated by mass differences between successive

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The identification of immunogenic compounds from *L. obliqua*'s bristle extract was performed on gel-based approach using the polyclonal horse anti-Lonomic hyperimmune serum and anti-Lopap specific rabbit serum produced by the Butantan Institute [9]. Bidimensional electro‐ phoresis of bristle extract revealed 157 silver stained spots, under non-reducing conditions (without DTT and iodoacetamide addition), providing an overview protein mapping (Figure 6A). However, 153 spots were immunodetected using anti-Lonomic serum (Figure 6B) and 30 spots detected using anti-Lopap serum (Figure 6C). Abundant proteins from 24 selected colloidal Coomassie Blue gel spots, corresponding to immunogenic proteins, were digested with trypsin and analysed by tandem mass spectrometry. The identification searches were carried out using the *L. obliqua* bristle EST databank. Lipocalins (spots 05, 09, 10, 14, 15, 16, 18, 24), cuticle protein (spots 05, 06, 07, 08, 11, 12, 13) and serpins (spot 21) were amongst the proteins identified (Figure 6A) [9]. Lipocalins can play a role in homeostasis and inflammation, as a defense mechanism in haematophagous arthropods. Lopap, characterized as a lipocalin protein member, and its all isoforms were highly represented as immunogenic proteins, revealed by the specific anti-Lopap serum (Figure 6C). The bristle' cDNA libraries also confirm the high abundance of lipocalins. As previously described [9, 19], these proteins have impor‐ tant role in envenoming. The cuticle proteins identified can be related to the inflammatory response caused by macerated spicule proteins. The serpin protein may also be involved in

Besides the biochemical and pharmacological tests, the quality control of serum and vaccine production can be monitored by proteomic technologies [64], such as chromatographic analyses, bidimensional electrophoresis and immunoblotting, once they are able to detect protein degradation and also confirm the presence of specific antibodies. However, immuno‐ therapy can be more effective if a better characterization of venom composition is performed, improving immunization procedures, increasing its specificity and reducing side effects. The new generation of high affinity antibodies against low abundant immunogenic toxins can be

A novel approach is the investigation of post-translational modifications (PTM) that affect antigen recognition, given that many peptides presented to T cells by the major histocompa‐ bility complex are post-translationally modified [66]. Glycosylation and phosphorylation are important PTMs of proteins, playing crucial roles in several biological processes, including cell recognition and signalling pathway [67, 68]. Some potential targets for cancer therapy are

Otherwise, phosphorylated proteins are usually enriched by immunoprecipitation (mainly for phosphotyrosine peptides) or by chromatographic procedures, such as Strong Cation eX‐ change (SCX), Hydrophilic Interaction Liquid Chromatography (HILIC), Immobilized Metal Affinity Chromatography (IMAC) or Metal Oxide Affinity Chromatography (MOAC).

peaks in the spectrum, which are related to the individual mass of their residues.

the defense mechanism.

Applications

184

evaluated by an antivenomic approach [64, 65].

based on glycosylated and phosphorylated epitopes discoveries [59].

**Figure 6.** Bidimensional electrophoresis and immunoblotting from *Lonomia obliqua*'s bristle extract. (A): silver stained bidimensional gel (100 μg of protein applied) under non-reducing condition. Panels (B) and (C): PVDF im‐ munoblotted 2D gels incubated with anti-Lonomic horse serum diluted 1:500 (B) or with anti-Lopap rabbit serum diluted 1:250 (C) [9].

Different metals may be used (iron, zirconium, gallium, etc) and peptides eluted by acidic or basic conditions, releasing mono-phosphorylated and multi-phosphorylated peptides, respectively [67].

The simultaneous screening of thousands of proteins from complex samples in a fast and sensitive manner can be performed using protein arrays. Amongst the different protein microarray applications are biomarker discovery, protein interaction studies, enzymesubstrate profiling, immunological profiling and vaccine development. As our interest is in the immune response, an antibody microarray can be used for identification of antigens that react specifically with the antibodies spotted on a solid support, with the complex formed then detected by fluorescence [59].

A large number of not yet identified proteins are considered as unknowns, but higher probabilities of identifications are reached when different methodologies are applied for analysis of complex samples. The combination of several proteomic techniques described here could improve the detection of immunogenic compounds and create new perspectives for effective immunotherapies.
