**2. Proteomic technologies for the identification of plasma proteins of early stage CRC**

The use of blood or plasma for screening or diagnosis of CRC is the most attractive non-invasive material available for the identification of clinically relevant protein biomarkers. Most commonly, candidate protein biomarkers of early stage CRC are identified using MS-based proteomics techniques. Below we list the limitations and advantages of the most common sample preparation and proteomics techniques specifically to identify candidate biomarkers in the plasma of early stage CRC. These techniques face a number of limiting factors, which have reduced the utility of proteins revealed by proteomics. Indeed, factors including the extreme dynamic range of proteins within plasma [28], the variability in collection and processing methods [21], preanalytical and analytic processes [29], and the inherent heterogeneity of patient samples [30], have all hindered uniform consent for which biomarkers are the most relevant for use in the setting of early stage disease.

As a small number of highly abundant proteins such as; albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, comprise 90% of the human plasma proteome [31], therefore little capacity is left for the identification of lower abundance proteins to be used as early stage markers of CRC [32]. Researchers have thus turned to immunodepletion strategies to enrich for low abundant proteins, resulting in a 25% increase in identified proteins and 4-fold increased enrichment of non-targeted plasma proteins using peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [31]. These pioneering studies have paved the way for high-resolution LC-MS/MS studies employed on depleted samples, routinely affording researchers with the capacity to identify 100 s if not 1000 s of plasma proteins during the course of a proteomics investigation [21] (**Figure 2**).

In the context of proteogenomic approaches to biomarker discovery [33], recent studies have also made some progress in reducing variability during collection and processing, revealing the suitability of human plasma proteins for qualitative and quantitative proteomic analysis after collection and storage for up to 48 hours at room temperature in cell free DNA-optimized blood collection tubes [21]. These tubes have been developed to overcome some of the issues that delays in processing time, temperature, and handling contribute to the deterioration of non-protein– based biomarkers [34] and now protein biomarkers [21]. Although not yet in widespread use, future studies may show that these cell stabilization tubes reduce plasma contamination by proteins originating from blood cells during collection and storage, thus increasing the reproducibility of proteomics-based biomarker discovery projects (**Figure 2**).

#### **2.1 Gel-based separation platforms**

Two-dimensional electrophoresis (2DE) coupled to mass spectrometry is a very accurate and sensitive method of large-scale protein separation using human CRC tissue [35]. The application of this preparative platform, which facilitates the resolution of protein mixtures on the basis of proteins isoelectric point and molecular weight has been extensively employed using CRC tissue [36–38]. These techniques can be combined with any analytical MS platform to identify changes in protein abundance between samples. Results of these studies are most commonly validated using orthogonal immunological-based techniques using plasma including; ELISA, flow cytometry, immunoblotting. Recently, two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was employed on early and late stage CRC plasma samples, identifying apolipoprotein A1 (APOA1) as a potential marker

*Advances in the Molecular Understanding of Colorectal Cancer*

undertake colonoscopy examination have an estimated 90% decreased incidence of colon cancer than those who do not [12]. Early detection of polypoid disease and

Over the last 2 decades, unprecedented technological advancement in proteinbased mass spectrometry (proteomics) has radically changed the landscape of biomarker research [13] (**Table 1**). This has facilitated the characterization of complex cellular proteomes [14–19], research that has identified hundreds of over and under expressed proteins in carcinoma patients using tumor tissue, histological sections, plasma or fecal samples when compared to matched normal tissues [20–24]. Despite this, with the exception of Carcinoembryonic antigen (CEA) and Cancer antigen 19-9 (CA 19-9) [25], no new protein biomarkers have made it into routine clinical practice [21, 26, 27]. In this book chapter, we have sought to present an overview of the diagnostic and prognostic protein biomarkers of early stage CRC to aid in the development of accommodating future screening tools that will continue to increase the rate at which early stage CRC is diagnosed and treated. We also review the use of contemporary proteomic approaches to address many of the long-standing challenges in the field of human CRC plasma proteomics and specu-

subsequent removal of polyps therein prevents progression to CRC [8].

late on the future clinical applications of these technologies (**Figure 2**).

*Overview, and advantages and disadvantages of using gel-free quantitative proteomic approaches for the* 

*identification of plasma protein biomarkers of early stage colorectal carcinoma.*

**132**

**Figure 2.**

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 stage CRC.
