**2.2 Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)**

SELDI-TOF MS also known as ProteinChip® technology, is a high-throughput technique that can purify and identify plasma protein biomarkers [41]. The method offers simplicity as proteins are bound to a solid-phase chromatographic surface, which helps protein isolation from crude mixtures, with non-bound proteins being washed away. The remaining bound proteins are mixed with an energy-absorbing matrix such as sinapinic acid (SPA) or α-cyano-4-hydroxycinnamic acid (CHCA) to induce ionization and desorption of the proteins on the surface of the plate. MALDI-TOF MS is then used to generate a unique mass-to-charge ratio (m/z) of the desorbed molecules, which are analyzed as they fly down the TOF tube and detected as peaks in a mass spectrum [42]. The normalized peak intensity is directly proportional to the concentration of the corresponding protein molecule in the sample.

One of the earliest reports of SELDI-TOF MS for the identification of early stage CRC plasma biomarkers identified a four protein peak m/z profile (m/z: 3191.5, 3262.9, 3396.3 and 5334.4) that was able to discriminate CRC from healthy controls with a sensitivity and specificity exceeding 90% [43]. Furthermore, two additional protein peaks (m/z: 9184.4 and 9340.9) were described as being able to discriminate plasma from patients with primary CRC from those with metastatic CRC [43]. In the same year, a similar study employing SELDI-TOF revealed a set of two protein peaks (m/z: 8132 and 4002) that could discriminate CRC from control again with >90% sensitivity and specificity [44]. This study was followed up some years later using an independent, patient case-control series of blood samples collected at multiple sites. However the latter study failed to discriminate plasma from CRC patients from healthy controls using these two protein peaks. Rather, the study identified two new protein peaks (m/z: 3961 and m/z 5200) in CRC plasma compared to healthy controls, again yielding very high sensitivity and specificity [45]. Drift and intensity of m/z were suggested to be responsible for the variation in reproducibility between the studies, an inherent limitation of SELDI-TOF based biomarker discovery projects, mostly underpinned by the wide dynamic range of human plasma.

#### **2.3 Chromatographic separation platforms**

While the analysis of intact proteins by 2DE is likely to continue to play an important role in comparative studies of the CRC tissue proteome, recent technical developments have heralded a new era in proteomics where the emphasis is placed on peptides rather than on whole proteins. Trypsin-based proteomics is now well recognized at the starting point in any proteomics investigation. Hydrolysis of

**135**

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection…*

peptide bonds in proteins is achieved using proteolytic enzymes resulting in the generation of an even more complex peptide mixture. However, the smaller size of peptides makes them much more homogenous structures than proteins. This coupled with the continued maturation of nanoscale chromatographic strategies, and the revolution of electrospray ionization MS (ESI-MS) [46] have meant that the rapid and detailed analysis of the human proteome using tryptic peptides is now

Tryptic digestion at the whole proteome level increases the complexity of a protein sample, therefore peptide purification techniques including reversedphase high performance liquid chromatography (RP-HPLC) are key for achieving increased sensitivity through flow separating eluting peptides entering the MS over time. RP-HPLC is most commonly used for one-dimensional (1D) peptide purification in proteomics. In RP-HPLC, peptides are generally retained due to hydrophobic interactions with the stationary silica phase. Polar mobile phases, such as water mixed with acetonitrile, are subsequently used to elute the bound peptides in order of decreasing polarity (increasing hydrophobicity). While reversed phase chromatography can be used as the sole separation procedure for moderately complex peptide mixtures prior to LC-MS/MS analysis, it is generally considered to have insufficient resolution for the analysis of more complex mixtures. This reflects the fact that although an MS instrument can perform mass measurements on several co-eluting peptides, if many peptides co-elute, the instrument cannot fragment them all and valuable information is likely to be irretrievably lost. Therefore, 2D-HPLC fractionation strategies including ion exchange chromatography (IEX), strong cation exchange (SCX), hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and hydrophilic strong anion exchange chromatography (hSAX) are commonly employed prior to RP-HPLC. These 2D approaches were used in the draft mapping of the human proteome [48, 49], and continue to be a key preparative step in the

common place in the MS laboratories of the world [47] (**Figure 2**).

successful application of whole proteome based investigations.

In addition to their utility in building an in-depth understanding of the CRC plasma proteome, gel-free strategies have also proven to be particularly amenable for use in comparative profiling applications. Indeed, since peptides are inherently less variable than their parent proteins, it has been argued that they constitute a more reliable basis for quantitative comparisons. This property has been exploited for the development of a suite of isobaric-tag based labeling strategies, which facilitate the simultaneous comparison of complex proteomic mixtures using different sample populations. The most common of these approaches used in plasma introduces a isobaric tag covalently bound to the N-terminus and side chain amines of plasma peptides [e.g. isobaric tags for relative and absolute quantitation (iTRAQ ) [50] and tandem mass tags (TMT)] [51]. Each of these approaches allows for relative quantification between samples based on the intensities of the reporter produced by precursor-ion fragmentation in the low m/z region of spectra. In each technique, the isobaric tags possess identical chemical properties to ensure similar behavior during chromatographic peptide separation and MS1 applications, but thereafter present as an easily distinguishable mass difference. As such, chromatographic separation platforms have become viable alternatives to 2DE for the differential analysis of complex protein mixtures [52, 53]. The MS operates in Data Dependent Acquisition (DDA) mode so that during each duty cycle the MS cycles

**2.4 Quantitative proteomics**

*2.4.1 Isobaric labeling*

*DOI: http://dx.doi.org/10.5772/intechopen.80942*

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*

peptide bonds in proteins is achieved using proteolytic enzymes resulting in the generation of an even more complex peptide mixture. However, the smaller size of peptides makes them much more homogenous structures than proteins. This coupled with the continued maturation of nanoscale chromatographic strategies, and the revolution of electrospray ionization MS (ESI-MS) [46] have meant that the rapid and detailed analysis of the human proteome using tryptic peptides is now common place in the MS laboratories of the world [47] (**Figure 2**).

Tryptic digestion at the whole proteome level increases the complexity of a protein sample, therefore peptide purification techniques including reversedphase high performance liquid chromatography (RP-HPLC) are key for achieving increased sensitivity through flow separating eluting peptides entering the MS over time. RP-HPLC is most commonly used for one-dimensional (1D) peptide purification in proteomics. In RP-HPLC, peptides are generally retained due to hydrophobic interactions with the stationary silica phase. Polar mobile phases, such as water mixed with acetonitrile, are subsequently used to elute the bound peptides in order of decreasing polarity (increasing hydrophobicity). While reversed phase chromatography can be used as the sole separation procedure for moderately complex peptide mixtures prior to LC-MS/MS analysis, it is generally considered to have insufficient resolution for the analysis of more complex mixtures. This reflects the fact that although an MS instrument can perform mass measurements on several co-eluting peptides, if many peptides co-elute, the instrument cannot fragment them all and valuable information is likely to be irretrievably lost. Therefore, 2D-HPLC fractionation strategies including ion exchange chromatography (IEX), strong cation exchange (SCX), hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and hydrophilic strong anion exchange chromatography (hSAX) are commonly employed prior to RP-HPLC. These 2D approaches were used in the draft mapping of the human proteome [48, 49], and continue to be a key preparative step in the successful application of whole proteome based investigations.

## **2.4 Quantitative proteomics**

#### *2.4.1 Isobaric labeling*

*Advances in the Molecular Understanding of Colorectal Cancer*

of early stage CRC [39]. Interestingly, this study also showed decreased levels of galectin-7 (GAL-7) in patients with early stage disease compared to healthy controls. CRC tissue examination of GAL-7 revealed 100% negative immunoreactivity implying that it may not might not be originating from the tumor tissues [39]. Gel-based separation approaches coupled to mass spectrometry face significant limitations related to their reproducibility, low sample number capacity, poor resolution of low abundant potential biomarker proteins, poor resolution of highly acidic/basic proteins and of proteins with extreme size or hydrophobicity, and co-migration of multiple proteins in a single spot that renders comparative quantification rather inaccurate [40]. Therefore, more recently researchers have largely focused on gel-free approaches for the identification of biomarkers of early

**2.2 Surface-enhanced laser desorption/ionization time-of-flight mass spectro-**

SELDI-TOF MS also known as ProteinChip® technology, is a high-throughput technique that can purify and identify plasma protein biomarkers [41]. The method offers simplicity as proteins are bound to a solid-phase chromatographic surface, which helps protein isolation from crude mixtures, with non-bound proteins being washed away. The remaining bound proteins are mixed with an energy-absorbing matrix such as sinapinic acid (SPA) or α-cyano-4-hydroxycinnamic acid (CHCA) to induce ionization and desorption of the proteins on the surface of the plate. MALDI-TOF MS is then used to generate a unique mass-to-charge ratio (m/z) of the desorbed molecules, which are analyzed as they fly down the TOF tube and detected as peaks in a mass spectrum [42]. The normalized peak intensity is directly proportional to the concentration of the corresponding protein molecule in the

One of the earliest reports of SELDI-TOF MS for the identification of early stage

CRC plasma biomarkers identified a four protein peak m/z profile (m/z: 3191.5, 3262.9, 3396.3 and 5334.4) that was able to discriminate CRC from healthy controls with a sensitivity and specificity exceeding 90% [43]. Furthermore, two additional protein peaks (m/z: 9184.4 and 9340.9) were described as being able to discriminate plasma from patients with primary CRC from those with metastatic CRC [43]. In the same year, a similar study employing SELDI-TOF revealed a set of two protein peaks (m/z: 8132 and 4002) that could discriminate CRC from control again with >90% sensitivity and specificity [44]. This study was followed up some years later using an independent, patient case-control series of blood samples collected at multiple sites. However the latter study failed to discriminate plasma from CRC patients from healthy controls using these two protein peaks. Rather, the study identified two new protein peaks (m/z: 3961 and m/z 5200) in CRC plasma compared to healthy controls, again yielding very high sensitivity and specificity [45]. Drift and intensity of m/z were suggested to be responsible for the variation in reproducibility between the studies, an inherent limitation of SELDI-TOF based biomarker discovery projects, mostly underpinned by the wide dynamic range of human plasma.

While the analysis of intact proteins by 2DE is likely to continue to play an important role in comparative studies of the CRC tissue proteome, recent technical developments have heralded a new era in proteomics where the emphasis is placed on peptides rather than on whole proteins. Trypsin-based proteomics is now well recognized at the starting point in any proteomics investigation. Hydrolysis of

**134**

stage CRC.

sample.

**metry (SELDI-TOF MS)**

**2.3 Chromatographic separation platforms**

In addition to their utility in building an in-depth understanding of the CRC plasma proteome, gel-free strategies have also proven to be particularly amenable for use in comparative profiling applications. Indeed, since peptides are inherently less variable than their parent proteins, it has been argued that they constitute a more reliable basis for quantitative comparisons. This property has been exploited for the development of a suite of isobaric-tag based labeling strategies, which facilitate the simultaneous comparison of complex proteomic mixtures using different sample populations. The most common of these approaches used in plasma introduces a isobaric tag covalently bound to the N-terminus and side chain amines of plasma peptides [e.g. isobaric tags for relative and absolute quantitation (iTRAQ ) [50] and tandem mass tags (TMT)] [51]. Each of these approaches allows for relative quantification between samples based on the intensities of the reporter produced by precursor-ion fragmentation in the low m/z region of spectra. In each technique, the isobaric tags possess identical chemical properties to ensure similar behavior during chromatographic peptide separation and MS1 applications, but thereafter present as an easily distinguishable mass difference. As such, chromatographic separation platforms have become viable alternatives to 2DE for the differential analysis of complex protein mixtures [52, 53]. The MS operates in Data Dependent Acquisition (DDA) mode so that during each duty cycle the MS cycles

through a short survey scan of the eluting peptides or precursor-ions, then a series of n (~10–15) MS2 scans, during which each of the precursor-ions are isolated, fragmented and their fragment-ions are detected. Database searching is then performed on the MS2 fragmentation spectra and used to identify the sequence of their MS1 parent peak. Limitations in this technology underpin some of the variation seen in MS based biomarker studies since MS2 spectra rarely allow unambiguous identification of the precursor-ions. Nevertheless, the application of quantitative DDA (iTRAQ ) to investigate a panel of 10 CRC plasma samples revealed Orosomucoid 2 (ORM2) to be elevated compared to 10 healthy control samples [54]. ORM2 expression was confirmed in CRC tissues compared with corresponding adjacent normal mucosa; however no significant association between ORM2 concentrations and TNM stage or histological grade was shown. Nevertheless, an interesting finding to arise from this work was that plasma levels of ORM2 were higher in patients with inflammatory bowel disease, than in patients presenting with either a normal colorectum, hyperplastic polyps, or adenoma [54]. Thus, ORM2 appears to function in modulating the activity of the immune system, potentially mediating escape from immune recognition; an important first step during transformation.

A recent study by our group assessed whether the plasma samples of CRC patients stored in specialized blood collection tubes (e.g., PAXgene or STRECK; referred to as "BCT"), designed to reduce plasma DNA (pDNA) contamination and enhance low-abundance DNA target detection, was amenable for comparative and quantitative proteomics [21]. Eight patients with Stage I–IIA, and one patient with Stage IIIB were collected pre- and post- resection, in both BCT and EDTA tubes, and subjected to comparative and quantitative analyses using TMT. Of the 641 unique proteins identified across all samples, 184 proteins showed ±0.5 log2 fold-change in peptide abundance pre- versus post-operation. Label-free targeted proteomics validation using parallel reaction monitoring (PRM, discussed below) showed the most well recognized blood marker of CRC, CEA, was significantly more abundant pre- compared to post-operation in patients with early stage disease when collected and stored in BCT prior to MS. The same trend was also seen for gelsolin (GSN), structural maintenance of chromosomes protein 1B (SMC1B), E3 ubiquitin-protein ligase SHPRH (SHPRH), and semaphorin-3C (SEMA3C), highlighting the importance of preanalytical considerations during biomarker investigations using proteomic-based techniques [21].

### *2.4.2 Label-free quantification*

Label-free mass spectrometry has recently emerged as a quantitative tool for the analysis of CRC plasma proteins. In the absence of isobaric-tagged based modifications, this rapid, low-cost technology relies on a workflow in which individual samples are analyzed (e.g. by LC-MS or LC-MS/MS) separately prior to protein quantitation via precursor ion (intact peptide) signal intensity or via spectral counting. The development of high-resolution accurate mass time-of-flight (TOF), and Orbitrap MS facilitates the extraction of precursor ion peaks at the MS1 level, permitting identification based at MS2 level (**Table 1**). The m/z ratios for all ions are detected and their signal intensities at a particular chromatographic retention time recorded. Owing to the tight correlation between signal intensity and ion concentration, relative peptide levels between samples can be determined directly from these peak intensities. Similarly, spectral counting exploits the strong correlation between protein abundance and the number of MS/MS spectra. This approach involves counting the number of peptide-specific spectra identified in different biological samples and the subsequent integration of these data for all measured peptides of the protein(s) that are quantified.

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*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection…*

Examples of the application of label-free based proteomic profiling in the context of CRC include comparative analyses of the plasma samples from a cohort of 118 CRC patients compared with 96 healthy controls [55]. This study reported the identification of 373 plasma proteins, with 69 linked to CRC. Of the 69 CRC associated proteins, 2 proteins; Macrophage mannose receptor 1 (MRC1) and S100A9, were verified as being upregulated in CRC by immunoblot analysis and proved effective in identifying CRC from healthy controls with high accuracy, using

Targeted proteomics, using multiple (MRM) (also known as; selected reaction monitoring or SRM) [56] or parallel reaction monitoring (PRM) [57] technologies enables absolute quantitation of multiple peptides per chromatographic experiment by exploiting the unique capabilities of triple quadrupole (QQQ ) and quadrupole Orbitrap MS and the unique characteristics of the targeted peptides. Analysis is performed by the acquisition of selected events across the chromatographic retention time, of predefined pairs of precursor-ion and product-ion masses for MRM, or individual precursor-ions for PRM. The technique becomes an absolute quantitation tool by spiking isotopically labeled synthetic peptide(s) into the complex sample of interest, which act as internal standards for any peptide(s) of interest. The labeled peptide standards are designed to mimic those generated by tryptic sample digestion, differing by only a few Daltons dependent on the isotopic label used. This enables endogenous and isotope-labeled peptides to be subjected to targeted MS/MS analysis and differentiated by the unique MS2 mass spectra provided by the isotopic label. MRM assay development and optimization are key elements for this method of targeted quantitation. This is somewhat mitigated using PRM-based targeted proteomics, owing to the high-resolution mass accuracy of quadruple Orbitrap MS for precursor-ion selection and the monitoring of all MS2

High-throughput targeted proteomics using MRM in immunodepleted blood plasma has previously been employed to measure the abundance of large numbers of candidate CRC plasma proteins using 137 [23] and 1045 [20] confirmed CRC patients. These powerful studies highlight the capabilities of current MS technologies. Indeed, no less than 187 and 392 candidate marker proteins were simultaneous monitored, respectively. These analyses have aided in the development of candidate panels of plasma protein markers that can be monitored simultaneously to identify

Sequential windowed acquisition of all theoretical fragment-ion mass spectra (SWATH-MS) is a quantitative MS approach heralded as among the most important recent developments in proteomics research [58]. Driven by the recent advances in speed and sensitivity of the new generation of high resolution Triple-TOF MS, these technologies afford the ability not only to determine which proteins are present in the proteome, but also to accurately quantitate without the need for label-based methods, or by limited numbers of targeted peptides. This is due to the lower duty cycle of a Triple-TOF MS compared to an Orbitrap-based mass analyzers [59]. SWATH-MS operates in Data Independent Acquisition (DIA) in which all ions within a selected m/z range are fragmented and analyzed in a second stage of tandem mass spectrometry. In combining the unique advantages of traditional DDA (high-throughput) and MRM (high reproducibility and consistency) technologies,

*2.4.3 Multiple and parallel reaction monitoring (MRM/PRM)*

fragment-ions used for quantitation in parallel.

CRC in the symptomatic population [20, 23].

*2.4.4 SWATH MS*

*DOI: http://dx.doi.org/10.5772/intechopen.80942*

ELISA analyses [55].

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*

Examples of the application of label-free based proteomic profiling in the context of CRC include comparative analyses of the plasma samples from a cohort of 118 CRC patients compared with 96 healthy controls [55]. This study reported the identification of 373 plasma proteins, with 69 linked to CRC. Of the 69 CRC associated proteins, 2 proteins; Macrophage mannose receptor 1 (MRC1) and S100A9, were verified as being upregulated in CRC by immunoblot analysis and proved effective in identifying CRC from healthy controls with high accuracy, using ELISA analyses [55].
