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

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 fragment-ions used for quantitation in parallel.

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 CRC in the symptomatic population [20, 23].

## *2.4.4 SWATH MS*

*Advances in the Molecular Understanding of Colorectal Cancer*

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

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

investigations using proteomic-based techniques [21].

peptides of the protein(s) that are quantified.

*2.4.2 Label-free quantification*

**136**

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,

SWATH-MS can be deployed for both discovery and quantitation of all detectable peptides present in complex biological samples.

SWATH-MS also affords the added advantage that it does not rely on prior knowledge of the precursor peptide ions, instead acquiring information in a DIA manner and thus avoiding laborious assay development. The SWATH-MS workflow involves two key steps beginning with the generation of a spectral library (e.g. via conventional LC-MS/MS) through which acquired peptides are identified. During this acquisition mode, the mass spectrometer is programed to step within 2–4 s cycles through a set of precursor acquisition windows covering the mass range accessible by a quadrupole mass analyzer and also that in which most tryptic peptide precursors should fall (400–1200 m/z). During each cycle, the mass spectrometer fragments peptide precursors and records a complete, high accuracy fragment ion spectrum for all precursors that elute on the chromatograph. This is then followed by acquisition of SWATH-MS data for each sample under analysis, interrogation and matching against the spectral library to identify peptides, and finally extraction of specific peptide ions to enable area-under-the-curve quantitation between samples.

The first SWATH-MS study of CRC plasma also simultaneously assessed protein biomarkers from pancreatic cancer, lung cancer, prostate cancer, and ovarian cancer, all from patients diagnosed with early forms of these diseases. This sophisticated study employed sample enrichment and subsequent detection of tissuespecific secreted protein profiles via SWATH-MS. These data were used to generate a digital representation of the proteins from within each plasma sample that could be queried for the presence and quantity of specific peptides using a targeted data analysis [60]. Tumor specific biomarkers were detected for individual cancer types, as well as a common biomarker Thrombospondin-1 (THBS1), which was significantly altered in the blood of four of five carcinomas (CRC, lung, prostate and ovarian) [61]. These ground breaking studies highlight the potential of the new generation of analytical MS techniques for the detection of early stage.
