Cancer Biomarkers

Special Issue: Review Article
Received 20 August 2010, Accepted 23 August 2010 Published online in Wiley Online Library: 6 November 2010
(wileyonlinelibrary.com) DOI 10.1002/bmc.1528
The discovery and validation of colorectal cancer biomarkers

Ching-Seng Ang,a,d Jason Phungc and Edouard C. Nice*ac
ABSTRACT: Colorectal cancer is currently the third most common malignancy in the world. Patients have excellent prognosis following surgical resection if their tumour is still localized at diagnosis. By contrast, once the tumour has started to metastasize, prognosis is much poorer. Accurate early detection can therefore signicantly reduce the mortality from this disease. However, current tests either lack the required sensitivity and selectivity or are costly and invasive. Improved biomarkers, or panels of biomarkers, are therefore urgently required. We have addressed current screening strategies and potential protein biomarkers that have been proposed.The role of both discovery and hypothesis-driven proteomics approaches for biomarker discovery and validation is discussed. Using such approaches we show how multiple reaction monitoring (MRM) can be successfully developed and used for quantitative multiplexed analysis of potential faecal biomarkers. Copyright 2010 John Wiley & Sons, Ltd. Keywords: Colorectal cancer; Multiple reactioning monitoring; Faecal proteomic; Biomarker
Introduction
Unfortunately colorectal cancer (CRC) is very common in the Western world and is a signicant cause of morbidity and mortality. A 6% lifetime risk of developing CRC (Davies et al., 2005) translates into it being the third most common malignancy in the world (Parkin et al., 2001). In Australia, it is the second most commonly diagnosed malignancy in both males and females, as well as the second highest cause of cancer-related death (Young, 2009). However, CRC is potentially curable if detected early. Patients treated by surgical resection of their tumour when the tumour is still localized to the colon or rectum (Dukes Stage A) have a 5 year survival rate of >90% (White and Miller, 2007). By stark contrast, patients with Dukes Stage D cancer at presentation, where the tumour has metastasized to other organs, have a 5 year survival rate of <10% (Altekruse et al., 2009; Etzioni et al., 2003; Jemal et al., 2008; Ramsoekh et al., 2007; Fig. 1). It is known that the pathogenesis of CRC is a progressive accumulation of mutations including genes such as the APC, KRAS, SMAD2/ SMAD4 and p53 (Fearon and Vogelstein, 1990). This typically results in a 510 latency period and oers a window of opportunity for eective screening (Davies et al., 2005; Jimenez et al., 2010). Despite this, there has been little change in the rate of survival in CRC over the last two decades, with more than 50% of patients having regional or distant metastasis at the time of presentation (Etzioni et al., 2003).
However, it is costly, requires highly trained sta, is invasive and requires uncomfortable bowel preparation. There is also a small but nite risk of morbidity and mortality attached to the procedure (Davies et al., 2005). Unfortunately, in countries where national colonoscopy screening is available, compliance has often been low (Hundt et al., 2007; Tonus et al., 2006). This has been reported to be due to the unpleasant nature of the bowel preparation, discomfort of the procedure, the need for sedation and patients embarrassment. Flexible sigmoidoscopy (Terdiman, 2005) is often used as an alternative to colonoscopy since it is a rapid proceedure, requires no sedation or overnight hospital stay and has a very low complication rate (Hassan et al., 2008). However, it only allows screening of the distal colon (Fig. 2) and hence misses tumours located in the transverse and ascending colon and the caecum. It has a reported sensitivity of 69% and specicity of 95% in determining the presence or absence of disease (Goodbrand, 2008).
* Correspondence to: E. C. Nice, Head, Clinical Biomarker Discovery and Validation, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: ed.nice@med.monash.edu.au
a
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Melbourne, Australia The University of Melbourne, Melbourne, Australia Monash University, Department of Biochemistry, Melbourne, Australia Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, Bundoora, Australia Abbreviations used: CRC, colorectal cancer; CCAP, colorectal cancer associated protein; CEA, carcinoembryonic antigen; CT, computed tomography; FOBT, faecal occult blood test; FS, exible sigmoidoscopy; IMAC, immobilized metal anity chromatography; MALDI, matrix-assisted laser desorption/ionization; MRM, multiple reaction monitoring; SELDI, surface enhanced laser desorption and ionization time-of-ight mass spectrometry; QQQ, triple quadrupole; SEC, size exclusion chromatography
Current Screening Methods

The early detection of CRC or the pre-cancerous lesions (adenomas) would signicantly reduce the burden this cancer places on the community. Current screening options include the faecal occult blood test (FOBT), exible sigmoidoscopy, colonoscopy and virtual CT scanning (Davies et al., 2005; Levin et al., 2003; Ramsoekh et al., 2007). Colonoscopy, which has a reported sensitivity of 97% and specicity of 98% (Davies et al., 2005), is the gold standard for early detection of CRC (Zolg and Langen, 2004).
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Copyright 2010 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2011; 25: 8299
The discovery and validation of colorectal cancer biomarkers CRC do not bleed continuously but rather intermittently with variable blood loss (Levin et al., 2003). Also, the accuracy of gFOBT is often aected by high levels of dietary haeme found in red meat and peroxidases present in fruit and vegetables (Ouyang et al., 2005), an eect that has been shown to last for up to 3 days (Feinberg et al., 1990). Antioxidants such as Vitamin C can interfere with the assay, resulting in false negatives while gastro-intestinal bleeding, which can be caused by medications such as aspirin, can result in a false-positive result (Greenwald, 2005). The antibody-based iFOBT is more specic for human blood and has a reported sensitivity of 52.6% and a specicity of 87.2% (Nakazato et al., 2006). It is also more likely to be specic for blood originating from the colorectum as blood from the gut degrades as it travels down the digestive tract (Potack and Itzkowitz, 2010). Hence, upper gastro-intestinal bleeding (due to, for example, gastritis, gastric ulcer, duodenal ulcer) is less likely to skew results. Since all positive FOBTs should be followed up with colonoscopy, the poor selectivity and sensitivity of the test puts an excessive burden on current colonoscopy services and subjects a large number of patients to unnecessary colonoscopy. There is therefore an urgent need to develop more sensitive, reliable and specic screening tests for early stage colon cancer when therapy is most likely to be eective.
Figure 1. Colorectal cancer 5 year survival rate based on the tumour location at diagnosis (SEER annual report 19982006 (Altekruse et al., 2009).
The use of computed tomographic (CT) colonography has been suggested as an alternative to endoscopy for the detection of colorectal cancer (Pickhardt et al., 2003). This method uses radiation-based imaging and various methods of image manipulation (multiplanar reconstructions, three-dimensional constructions) to visualize the endolumen of the colon for polyps. Detection of large polyps (>10 mm) in populations with a higher prevalence of polyps has been reported to have a sensitivity of 90% and a specicity of 94%. In contrast, it has also been shown that, for CT colonography used in populations with average risk of development CRC and low-prevalence of polyps (i.e. the general population), sensitivity ranged from 55 to 94% at a specicity of ~95%. Furthermore, it is recognized that CT colongraphy has diculty in detecting at or depressed polyps (van Gelder et al., 2005). The procedure still requires prior bowel preparation and there are risks associated with the use of the ionizing radiation doses required to produce the images. Patients have also reported discomfort associated with the ination of the colon; these patients were not provided with bowel relaxants. Whilst patient compliance for CT colongraphy is better than for exible sigmoidoscopy (FS) or FOBT, attendance rates are still low at 28.4% (Edwards et al., 2004). The FOBT is currently the most widely prescribed primary screening tool. This test screens for the presence of blood or blood products in stool samples, is cheap and non-invasive, but has a high rate of both false-positive and false-negative results (Davies et al., 2005). The FOBT exists in two alternative assay forms, guaiac-based (gFOBT, e.g. Hemoccult II and Hemoccult II SENSA). which is dependent on the peroxidase-like activity of haem in the stool, and immunochemical-based tests (iFOBT, e.g. HemeSelect and InSure), which use human heamoglobin-specic antibodies. Four large randomized controlled trials have consistently shown that biennial screening with FOBT reduces CRC mortality (Kewenter et al., 1994; Kronborg et al., 2004; Scholeeld et al., 2002). A meta-analysis of these studies showed a 16% reduction in CRC mortality, or a 25% reduction when adjusted for non-compliance (Hewitson et al., 2007). However, a number of factors inuence the eectiveness of FOBT with many studies reporting low predictive values (Hewitson et al., 2007). Firstly,
Biological Samples for CRC Biomarker Discovery

A range of biological samples can be used as a starting point for CRC biomarker discovery (Jimenez et al., 2010). Historically cell lines have been used for a number of studies because they can be readily obtained and reproducibly cultured to generate reasonable levels of starting material. However one drawback for colon studies is that the corresponding normal tissue is dicult to maintain in culture and hence is not so readily available (Whitehead and Robinson, 2009). Additionally, most available cell lines have been derived from malignant tumours and hence are not a reliable model for early stage disease. In the case of CRC the Apc/Min mouse oers an excellent disease model relevant to the human situation. The C57BL/ ApcMin+ mice carry a mutant tumour suppressor gene which encodes a nonsense mutation at codon 850 of Apc. This mutation leads to truncation of the Apc protein, causing a disregulation in the Wnt/b-catenein signalling pathway (Moser et al., 1990; Su et al., 1992), similar to that observed in many human CRC (Nagase and Nakamura, 1993). The use of patient tissue samples allows comparison of the tumour sample with adjacent non-involved tissue. However sample sizes are frequently limiting as priority has to be given to the use of surgically obtained tissue for conventional diagnosis. It will be interesting to see whether MALDI imaging (Franck et al., 2009; Reyzer and Caprioli, 2007), where direct proling of proteins in tissue sections can be obtained, will prove complimentary to immunostaining or histological analysis (Ikonomou et al., 2009). The use of blood, which is minimally invasive, for routine clinical pathology is well accepted. However, its use in MS-based proteomics studies has not proved trivial. This is largely due to the fact that blood bathes all the organs of the body, and hence biomarkers arising from the tumour form only a very small subset of the total protein composition which, coupled with the wide dynamic concentration range spanning more than 10 orders of magnitude (Anderson and Anderson, 2002) and the presence of a
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CS. Ang et al.
Figure 2. testing.
Regions of the colorectum screened by dierent CRC diagnostic tests: FOBT, DNA stool testing, colonoscopy, CT, FS and proteomic stool
number of a small number of highly abundant housekeeping proteins, means only the most abundant tumour-derived proteins will be detected without signicant sample workup. Urine, which is readily available, has been primarily used for metabolomic studies (Cai et al., 2006; Qiu et al., 2010). Low-molecular weight proteins (less than 40 kDa) readily pass through the glomerular ltration barrier and are reabsorbed. Because of their low plasma concentrations, only small amounts of these proteins are seen in the urine. Stool testing provides important advantages over other screening methods. It is noninvasive, requires no unpleasant bowel preparations, can be performed at home and thus does not require time away to visit health care practitioners (Osborn and Ahlquist, 2005). One is not sample limited and importantly the stool eectively monitors the entire colon during transit down the gastro-intestinal tract (Osborn and Ahlquist, 2005). On the downside, there is reluctance by many people to handle faeces, leading to low compliance. Additionally, the gut microora can potentially play a major role in proteolysis in the human colon, leading to protein degredation (Gibson et al., 1989; Macfarlane et al., 1986). Lastly there is signicant variation in sample size and composition. Thus, as in urine where normalization based upon creatinine and other parameters is frequently used (Warrack et al., 2009), there is a need to nd a suitable control faecal marker to compensate for this. Faecal DNA testing is an alternative noninvasive CRC screening tool which relies on quantitatively assaying mutant DNA from stool samples (Ahlquist, 2009). Unlike occult blood, human DNA is shed continuously into the gut lumen but represents only 0.01% of total stool DNA (Potack and Itzkowitz, 2010). Initial studies have been able to identify presence of mutated KRAS and p53 genes in stool, both known to be associated with the pathogenesis of CRC (Ahlquist, 2009; Fearon and Vogelstein, 1990). Since then, second-generation DNA stool tests have combined a number of mutant DNA markers in a single test and have demonstrated better sensitivity over FOBT (Ahlquist, 2009; Imperiale et al., 2004). One study looking at 15 dierent markers on a single
panel showed a sensitivity of 92% for CRC and 82% for large adenomas, at a specicity of 93% (Ahlquist, 2009). However, despite promising initial results, DNA stool testing has been reported to have a number of problems including poor discriminative ability of the markers, lack of eective quantitative assaying techniques and bacterial degradation of the DNA during sample transportation (Ahlquist et al., 2008). Development of better genetic markers, DNA stabilizing buers and improved sample extraction techniques is underway and promises to increase the accuracy of future tests (Ahlquist, 2009). However, there are several signicant limitations restricting the use of DNA stool testing as a routine population-based screening tool. Firstly, the use of multi-marker panels makes the test very expensive and time-consuming to process (Davies et al., 2005). Pricing of single tests has been quoted at US$400800 (Davies et al., 2005; Levin et al., 2003) and lack of automation in processing samples makes DNA stool testing inappropriate on a population-level. Also, the test currently requires the entire bowel moment and the collection kit is large, revealing issues of patient acceptability and sample transport and storage (Levin et al., 2003).
Faecal Protein Biomarkers for CRC Screening

Analysis of tumour-derived faecal proteins or peptides provide a noninvasive, specic and sensitive means to detect CRC (Osborn and Ahlquist, 2005). Importantly, these proteins may be relatively more abundant in faeces than in blood (Kim et al., 2003). A number of proteins found in faeces have been proposed to be associated with the presence of CRC. These proteins reach the lumen and pass into faeces through three dierent mechanisms: leakage, secretion and exfoliation (Ahlquist, 2009; Osborn and Ahlquist, 2005). Proteins leaked into the lumen have been shown to be associated with CRC, e.g. haemoglobin, calprotectin (Aadland and Fagerhol, 2002), lactoferrin (Hirata et al., 2007), lysozyme (Dubrow and Yannielli, 1992), albumin (Nakayama et al., 1987) and alpha-1-antitrypsin (Dubrow and Yannielli, 1992).
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The discovery and validation of colorectal cancer biomarkers However, these markers have been found to be poor markers of CRC due to their low sensitivity and specicity when used individually. Secreted proteins potentially oer a much better source of potential biomarkers for screening as they can be analysed by relatively non-invasive techniques (Ouyang et al., 2005). For example mucus is normally secreted into the colorectum to lubricate and facilitate faecal movement through the digestive tract (Osborn and Ahlquist, 2005). The mucins, which are large (generally >200 kDa) proteins with predominantly O-linked oligosaccharide side chains (Kim et al., 1996), found within mucus have been shown to be dysregulated in neoplasia (Boland et al., 1982). Abnormal mucins have been hypothesized to be potential biomarkers for CRC (Byrd and Bresalier, 2004; Kim et al., 1996; Taylor et al., 2009). Other markers have been evaluated for potential use as biomarkers. Carcinoembryonic antigen (CEA), a serum protein marker used in monitoring of CRC, has recently been studied in faeces. One study showed faecal CEA to have better sensitivity for CRC than FOBT at 86% with a specicity of >90% (Kim et al., 2003). Levels of faecal CEA, measured using an automated immunoassay system, were 44.1 70.1 ng/mg stool in cancer patients and 3.7 3.5 ng/mg stool in controls. Minichromosomal maintenance protein (MCM2) and decay-accelerating factor (CAF) are two other faecal proteins that have been proposed for use as a screening tool. However, data to date have shown suboptimal sensitivities (Davies et al., 2002). Faecal tumour M2 pyruvate kinase (M2-PK) has also been suggested as a potential CRC biomarker (Tonus et al., 2006). This study measured samples from 33 patients with CRC, 21 patients with rectal carcinoma and 42 controls using a commercially available sandwich ELISA which specically detects the dimeric, cancer-related, form of the protein. The overall sensitivity for CRC was 78% at 93% selectivity. Levels correlated well with tumour stage. A more recent study by Karl et al. (2008) investigated six dierent faecal markers. The best individual diagnostic performance was for S100A12 followed by TIMP-1, haemoglobinhaptoglobin, haemoglobin, calprotectin and carcinoembryogenic antigen. Using a combination of markers, they showed a specicity and sensitivity of 98 and 79%, respectively, using the combination of haemoglobin haptoglobin and S100A12. The specicity and sensitivity were increased to 98 and 82% when TIMP-1 was added to the panel. Based on the clinical samples screened (186 CRC, 113 advanced adenoma and 252 control patients), this biomarker panel was found to detect CRC at a signicantly higher rate than iFOBT alone. The sensitivity for early stage cancer was 74%. RNA editing (Keegan et al., 2001; Maniatis and Tasic, 2002) and proteolytic processing (Ehrmann and Clausen, 2004). Moreover protein subspecies can be greatly increased through posttranslational modications such as phosphorylation, glycosylation, methylation and ubiquitination to name a few (Reviewed by Walsh et al. (Walsh et al., 2005)). Thus, while it is now generally accepted that the human genome only contains around 2025,000 proteins (IHGSC, 2004), the human proteome is predicted to be considerably higher (>150,000 individual proteins) (Hoehenwarter et al., 2010). It therefore represents a signicant analytical problem.
The Need for Sample Pre-fractionation to Mine Deeper into the Proteome
The critical factor for a successful mass spectrometry-based biomarker discovery study is the identication and quantication of all the proteins of interest. There is currently, however, no perfect or universal method for identifying the entire proteome or any instrument that is capable of identifying and quantifying the components of a complex protein sample in a simple onestep procedure (Nice et al., 2007). For example, in a large-scale identication of proteins using 2D-PAGE gel electrophoresis, geLCMS and 2D-LCMS, a total of 1218 proteins were identied by the three methods in exponentially growing Bacillus subtilis cells, but only 140 proteins were consistently identied in all three analyses (Wol et al., 2006). This study clearly demonstrated the need for orthogonal purication strategies that utilize the specic selectivities associated with each method. A large variety of alternative strategies for analysing the complexity of the proteome have been investigated. They can be broadly grouped into three main types: gel based techniques [e.g. 2D PAGE (Gorg et al., 2000; OFarrell, 1975), geLCMS (Li et al., 2003), chromatographic fractionation of proteins/peptides including multidimensional/ orthogonal liquid fractionation (Nice et al., 2007; Wang et al., 2002)], removal of abundant proteins or selective enrichment of proteins/peptides [e.g. abundant protein depletion (Gong et al., 2006), IMAC columns (Andersson and Porath, 1986), ICAT (Gygi et al., 1999), glycoprotein enrichment (Taylor et al., 2009; Yang and Hancock, 2004)] and advances in instrumentation [e.g. LTQ Orbitraps (Olsen et al., 2009), application of electron transfer dissociation (Syka et al., 2004) etc.]. Our own data (Ang et al., 2009; Nice et al., 2007) has also shown that alternative separation protocols can reveal the presence of dierent proteins in the same sample (see below). However, for such techniques to be ultimately suitable for routine clinical analysis they need to be capable of simple automation. In particular the use of magnetic-bead based supports appears to have signicant potential for this purpose (Safarik and Safarikova, 2004): they can be used with very crude, particulate, samples (unlike chromatographic supports or membranes), multiple washings can eliminate non-specic binding and, importantly, analysis can be readily automated using simple robotics (Nice et al., 2007; Rothacker et al., 2007; Safarik and Safarikova, 2004). Using a magnetic bead-based protocol we have been able to isolate exfoliated cancer cells, and measure the associated telomerase levels using luminescence-based detection, from the urine of bladder cancer patients and exfoliated colonocytes isolated from faecal samples from colorectal cancer patients postcolonoscopy and prior to resection (Rothacker et al., 2007). The potential of magnetic-bead based purication using supports with chromatographic functionality has recently been shown for
Proteomics in Colorectal Cancer Biomarker Discovery

The continuing search for improved biomarkers or panels of biomarkers has stimulated many groups to use proteomic or genomic-based approaches for their discovery. Proteomics is the large-scale study of proteins (both qualitative and quantitative) to comprehensively map biological processes (Anderson and Anderson, 1998; Wilkins et al., 1996). In all organisms, the proteome is more complex than the genome for several reasons. The proteome is dynamic compared with the relative stable genome, with numerous tissues and organelles within the organisms each with individual developmental and temporal specicities. Many dierent proteins can be produced from a single coding sequence due to cellular events such as alternative transcriptional initiation (Mitchell and Tjian, 1989), alternative splicing,
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CS. Ang et al. expression proling and biomarker discovery using samples from patients with head and neck cancer pre- and post-treatment (Freed et al., 2008). Bruker Daltronics have released a range of magnetic-bead based supports (www.bdal.com, ClinProt) with various selectivities including reversed-phase, ion exchange, IMAC and lectin anity. More recently automated SPE using robotic control of chromatographic supports loaded into pipette tip-like columns (Phynexus, Atoll) has also been reported (www. tecan.com, application notes 396241, 395627, 395450). These include, for example, the identication of dierentially expressed proteins in serum (Engwegen et al., 2008; Liu et al., 2006a, b; Ward et al., 2006), the use of cancer cell lines (Zhao et al., 2007), comparison of the secretome of colorectal cancer cell lines (Mathivanan et al., 2010; Wu et al., 2008), comparison of urine samples (Ward et al., 2008) or comparison of paired tumouradjacent normal colorectal tissues (Friedman et al., 2004; Ma et al., 2009; Roessler et al., 2006). Proling of blood, tissues and the secretome from cell lines will be reviewed in the following sections. It cannot be overstated that for all proteomics studies where comparative studies are to be made, standard operating procedures must to be generated, validated and strictly adhered to (Apweiler et al., 2009). The HUPO Plasma Proteome Project has addressed these issues for plasma proteome samples (Rai et al., 2005). Several recent editorials have discussed the issue of bias in clinical biomarker studies on sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary eorts involving clinicians and scientists (Diamandis, 2007; Hortin, 2005).
Comparative Proteomics Strategies for the Discovery of CRC Biomarkers

The use of comparative proteomics (i.e. analysis of the changes to the proteome between the diseased and non-diseased state) is fundamental in the discovery of potential cancer biomarkers (proteomics, peptidomics, metabolomics). The traditional approach to comparative proteomics involves running samples from two dierent conditions (i.e. cancer and normal) on multiple 2D-PAGE gels, staining each gel with a protein stain, imaging the gels, then overlaying and aligning the two resultant images using sophisticated image analysis software. After comparative analysis of the spot intensities, spots of interest which are dierentially regulated are excised, subjected to in-gel digestion with trypsin followed by spotting onto a MALDI plate to generate a peptide mass ngerprint for identication using a MALDI mass spectrometer. A second approach is based on stable isotope labeling of protein or peptides followed by MS and MS/MS. Basically, proteins and peptides in one of the samples are modied with an isotype tag. The two samples are then combined before being processed and analysed by mass spectrometry. Although the physical characteristics of the peptides remain the same, the masses of the isotopically labelled peptides are proportionally shifted in the spectrum, and the ratio between the intensities of the dierentially labelled peptides peaks then permits accurate relative quantitation of the proteins. Stable isotopes can be incorporated onto proteins using techniques such as the isotope coded anity Tag (ICAT; Gygi et al., 1999) and isotope coded protein labeling (ICPL; Schmidt et al., 2005). Incorporation into peptides involves techniques such as 18O proteolytic labeling (Yao et al., 2003), isobaric tags for relative and absolute quantication (iTRAQ; Ross et al., 2004) or other chemical labeling such as acetylation (Ji et al., 2000) and reductive amidation (Hsu et al., 2003). Stable isotopes can also be incorporated into growing cells using techniques like isotope labeling by amino acids in cell culture (SILAC; Ong et al., 2002) or in vivo metabolic labeling using inorganic ammonium and nitrate salts enriched in 15N (Krijgsveld et al., 2003). The third approach is the use of label free methodology (Zhu et al., 2010). Researchers are increasingly turning to label-free proteomics techniques for faster, cleaner and simpler results with limited sample manipulation. involving techniques such as comparison of aggregate ion intensities (Chelius and Bondarenko, 2002) or spectral counting (Liu et al., 2004). Coupled with these technologies has been the development of new bioinformatics tools that aid in automated label-free analysis for comparative LC-MS. Another label-free technique is the use of multiple reaction monitoring methodology (Gerber et al., 2003). This is discussed in detail below. There have been a number of studies using comparative proteomics strategies for discovery of colorectal cancer biomarkers.
Blood Proling
Owing to the sample complexity, a number of blood-based proling studies for colorectal cancer biomarker discovery have been based on the surface enhanced laser desorption and ionization time-of-ight mass spectrometry (SELDI) approach. This technique involves incubating the protein mixture on a protein chip array with dened chemical or biochemical functionality (e.g. reversed phase, ion-exchange, IMAC, antibody anity). Proteins that have anity to these surfaces will be enriched and retained and can be subsequently characterized by TOF mass spectrometry (Hutchens and Yip, 1993). In a study by Ward et al. (2006), a total of 62 CRC and 31 non-cancer serums were screened by SELDI and the results subjected to articial neural network analysis based on the altered intensities of a number of characteristic peaks. This resulted in a reported assay sensitivity and specicity for CRC of 95 and 91%, respectively. Three of the dierentially regulated peaks were subsequently identied to be complement C3a des-arg, a1-antitrypsin and transferrin (Ward et al., 2006). A similar SELDI approach was used to analyse serum from74 CRC patients and 48 age- and sex-matched healthy subjects to identify a diagnostic pattern for the identication of CRC samples (Liu et al., 2006b). Using the dierential proteomic patterns, sensitivity and specicity of 95% was achieved in an independent set of masked serum samples from 60 CRC patients and 39 healthy subjects, although the identity of the representative peaks was not determined. A third study using the SELDI approach used a training set of 63 patients with CRC, 20 with benign colorectal diseases and 26 healthy volunteers (Zheng et al., 2006). Using a four-peak model, they were able to achieve a sensitivity and specicity of 87.5 and 93.8% respectively in discriminating cancer from non-cancer samples. However, as in the Liu et al. (2006b) study, the identity of the four discriminating peaks was not reported. Much of the early SELDI data has attracted signicant criticism with valid questions being raised as to experimental design, lack of reproducibility, statistical processing of the data, the low resolution of the mass spectrometer and the biological relevance of the generated data (Baggerly et al., 2004; Ivanov et al., 2006; Lambert et al., 2005; Paweletz et al., 2006; Weston and Hood, 2004). In many cases, including some of the examples cited above, peak patterns have been used as diagnostic ngerprints
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The discovery and validation of colorectal cancer biomarkers without characterization of the individual proteins. In some studies, when the putative biomarkers were fully characterized they were found to not be directly associated with tumour biochemistry, but instead correspond to ubiquitous proteins, such as the family of acute-phase response proteins (Villar-Garea et al., 2007; Engwegen et al., 2006). Without some form of additional sample pre-fractionation and concentration trace components are unlikely to be detected. Although the technique was originally reported to be compatible with the analysis of high molecular weight compounds (up to 200 kDa), it appears to work optimally in the 1.520 kDa range (Seibert et al., 2005). Despite this, it is worth mentioning that in September 2009 the FDA approved the rst diagnostic tool (OVA1) based on biomarkers discovered and validated using the SELDI approach. This test uses a ve-biomarker panel: transthyretin (TT or prealbumin), apolipoprotein A-1 (Apo A-1), beta2-microglobulin (Beta2M), transferrin (Tfr) and cancer antigen 125 (CA 125 II) to determine the likelihood of ovarian cancer in women with pelvic mass for whom surgery is planned.
Cell Line Proling (Secretome Analysis)

The secretome denes all the proteins secreted by a cell, and can encompass diverse functions ranging from immunoregulatory to pathological processes including cancer invasion and metastasis (reviewed by Pavlou and Diamandis, 2010; and Hathout, 2007). Tissues or biological uids that are in contact with the tumour may contain high levels of these secreted disease-related proteins. Such an analysis has the advantage of directly studying the individual subcellular components that are responsible for the observed markers in biological uids. One of the most comprehensive analyses of the cancer secretome involved the study of 21 cancer cell lines derived from 12 cancer types (Wu et al., 2008). The secretome of these cancer cell lines were rst separated on SDS-PAGE followed by identication using MALDI-TOFTOF. In total 795 proteins were identied and CRMP-2, a protein involved in cell dierentiation and microtubule assembly (Fukata et al., 2002), was selected for screening in plasma samples by uorimetric competitive ELISA. A total of 201 normal and CRC patients were screened for the presence of CRMP-2, resulting in a sensitivity and specicity of 60.5 and 65.2%, respectively. In a more recent comparative analysis, the secretome of a primary colorectal cancer cell line (SW480) was compared with that of the corresponding lymph node metastatic cell line (Xue et al., 2010). A total of 910 proteins were identied of which 145 proteins were dierentially expressed. ELISA assays were developed for two of the dierentially regulated proteins: trefoil factor 3 and growth/dierentiation factor 15 (GDF15). They were demonstrated to be potential biomarkers for the prediction of colorectal cancer metastasis from measurement of their levels in 156 healthy controls and 144 CRC patients. Using a cuto value of 1144 pg/mL, GDF15 had a sensitivity and specicity of 77.8 and 99.4%, respectively in detecting CRC. A cuto value of 1323 ng/mL for trefoil factor 3 resulted in a sensitivity and specicity of 53.5 and 97.4%, respectively. Exosomes are a subset of the secretome which have recently received considerable attention. Exosomes are 40100 nm diameter nanovesicles that are released from dierent cell types (Pan et al., 1985). Studies have demonstrated the release of exosomes from tumour cells and transfer of tumour antigens to dendritic cells (Wolfers et al., 2001). Mathivanan et al. (2010) used the A33 antibody, which recognizes a membrane antigen that is expressed in normal human colonic and small bowel epithelium and more than 95% of human colon cancers but is not present on most other human tissues and tumour types (Catimel et al., 1996; Heath et al., 1997), to immunopurify colorectal cancer cell linederived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which almost 30% contained signal peptides, many of them potential colorectal cancer markers [e.g. A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), epidermal growth factor receptor, and ephrin-B1 and -B2; Mathivanan et al., 2010)].
Tissue Proling
Comparison of the proteome changes between tumour and neighbouring normal colorectal mucosa is a common methodology employed to identify proteins that have altered abundance associated with tumourigenesis. These protein tumour markers could potentially escape from the tumour and be detected in blood or faeces and hence form the basis for detection of the disease. One of the earlier studies in colorectal marker discovery by Friedman and colleagues using 2D PAGE based analysis (Friedman et al., 2004) identied a total of 52 unique proteins that had altered expression between the tumour and adjacent normal mucosa. A similar study carried out by Ma et al. (2009) identied 42 proteins that also had dierential expression; interestingly several proteins such as nucleoside diphosphate kinase, desmin, tropomyosin, myosin, transgelin and annexin were common to both studies. In particular, desmin, an intermediate lament protein identiable in the serum of CRC patients was shown to correlate with both severity of CRC and also as a prognostic marker based on screening of 92 CRC patients, 25 patients with benign bowel disease and 45 healthy controls (Ma et al., 2009). Roessler et al. (2006) used a 2D PAGE approach to compare the protein prole of colon tumour tissue, adjacent normal tissue and adjacent stripped mucosa from 16 patients. A tumour-associated antiapoptotic factor PSME3 (Nikaido et al., 1989) was detected in seven of the 10 tumour tissues but not in adjacent normal tissues (Roessler et al., 2006). Interestingly, dierential expression was not obvious on the observed spot pattern on 2D-SDS PAGE. The PSME3-containing spot on tumour gels showed no visible dierence to the corresponding spot on matched control gels. Subsequently MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in the same spot on tumour gels, whereas the matched spot on control gels contained only ANXA4. Screening of PSME3 was subsequently undertaken on 109 CRC and 317 healthy control samples and compared with the equivalent data for CEA. Receiver-operating characteristic curves were derived by plotting the relationship between the specicity and the sensitivity at various cuto levels. The area under the curve was 0.76 for CEA and 0.77 for PSME3.
Quantitative Biomarker Measurement

As shown by many of the examples presented above, antibodybased methods are currently the most widely used for quantitative biomarker measurement, using techniques such as ELISA, IHC or microarrays. Using Luminex uorescently-tagged microspheres, multiplexed antibody-based assays can be readily generated (de Jager et al., 2003) Whilst antibody specicity has been long thought by many to be unquestionable, a number of recent
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Figure 3. Schematic of protein biomarker identication and quantitation by MRM (left panel). Shotgun proteomics is based on tryptic digestion of the protein followed by prefractionation of the peptides on nano-reversed phase liquid chromatography. The characteristic mass of the individual peptides is then determined by ESI-MS. The peptides are fragmented (MS/MS) and the resultant fragment mass and fragmentation pattern used to search a sequence database to match the peptide sequence. (Right panel) Multiple reaction monitoring (MRM) works by selecting a known precursor mass and the signature fragmentation ions characteristic of a known peptide. In the example shown, at the rst quadrupole (Q1) only peptides with the mass of 769.9 were allowed through to the collision cell (Q2). After fragmentation of the peptide, fragment ions with mass of 958.5 (characteristic of the y9 ion) are allowed through to be specically detected. Multiple MRM cycles targeting several fragmentation ions of the parent peptide increase the condence of identication.
studies using proteomics and protein arrays (Haab and Zhou, 2004; MacBeath, 2002; Smolec et al., 2005) have demonstrated that antibody specicity is often lacking, and considerable care must be taken in validation of the signals observed (Ackermann and Berna, 2007; Carney et al., 2003; Michaud et al., 2003; Nice et al., 2007; Nishizuka et al., 2003; Smolec et al., 2005). The number of inconsistent, and sometimes contradictory, publications in this area provides ample evidence of this problem. In one publication (Michaud et al., 2003) a polyclonal antibody was found to cross react with over 1500 dierent proteins. A comparison of the EGFR levels in clinical samples using two dierent antibody-based assays (Baron et al., 2001) gave over a 1000-fold dierence in recorded levels, with no direct correlation between the two assay sets, clearly indicating a major problem with one (or both) of the assays. Additionally, homogeneous protein standards need to be carefully generated, validated and quantied for antibody-based assays (Barker, 2003; Smolec et al., 2005). The expense (estimated at US$1,000,000) (Stahl-Zeng et al., 2007) and time (13 years; Fortin et al., 2009)) of developing a validated ELISA coupled with recent advances in MS technology is further stimulating the development of quantitative MS technologies for protein and peptide biomarker analysis (Ackermann and Berna, 2007). The limited specicity of many ELISAs, and in many cases limited antibody availability, coupled with recent advances in MS
technology, is stimulating the development of quantitative MS technologies for protein and peptide analysis (Anderson and Hunter, 2006; Kuhn et al., 2004; Wolf-Yadlin et al., 2007). Indeed MS of small molecules is already routinely used for clinical assays including multiple analyte screening for inborn errors of metabolism (Dooley, 2003; Kuhara, 2007) and drug analysis (Streit et al., 2002). For proteins, multiple reaction monitoring (MRM, also known as selective reaction monitoring, SRM), in which a triple quadrupole mass spectrometer (QQQ) is used to monitor both the intact peptide mass and one or more specic fragment ions of that peptide over the course of an LC-MS experiment, is rapidly becoming the method of choice (Fig. 3) (Ackermann and Berna, 2007; Stahl-Zeng et al., 2007; Wolf-Yadlin et al., 2007). The relative advantages and disadvantages of antibody and MS-based techniques for quantitative analysis are shown in Table 1. Triple Quad-based MRM Quantitation The principals of this technology are shown in Fig. 3. Triplequadrupole mass spectrometers can be programmed to scan specic preset mass-charge (m/z) ratios thus oering a high duty cycle facilitating identication and quantication of low-level components in complex biological samples such as plasma, urine or faeces. Importantly, the use of MRM can reveal trace peptides present in complex biological samples (Fig. 4). The combination
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Table 1. Advantages and disadvantages of antibody and MS-based quantitative analysis Antibody based assays Disadvantages Development is slow and expensive (Stahl-Zeng et al., 2007) Antibodies need to be available Many antibodies lack specicity Epitopes needs to be preserved in the sample Multiplexing requires individual reagents to be developed for each target MS based assays Advantages Generic method Lends itself to multiplexing Multiple peptide signatures Specic epitope not required In established clinical use for small molecules (e.g. drugs of abuse, inborn errors of metabolism; Rashed et al., 1997) Medium throughput Low cost per sample Disadvantages Less sensitive than ELISA (ng/mL becoming routine; pg/mL requires additional fractionation) Sophisticated instrumentation required
Advantages
Once developed, can be applied to a large number of samples Sensitive (pg to ng/mL) Simple instrumentation Established technology High throughput Used for routine clinical analysis
Figure 4. The specicity of MRM for identifying specic proteins in a complex sample. Faecal proteins were rst prefractionated using RP-HPLC and an individual fraction selected for futher analysis. Following tryptic digestion, the generated peptides were monitored on a mass spectrometer using both full scan (total ion chromatogram, TIC) and MRM mode. In the MRM mode, haemoglobin, haptoglobin and CEA were specically monitored, allowing them to be readily identied in the complex mixture (collaboration with Dr Alun Jones, University of Queensland, Australia).
of retention time, peptide mass and fragment mass practically eliminates ambiguities in peptide assignments and extends the quantication range to 45 orders of magnitude with sensitivity for direct analysis of 50100 ng/mL which can be extended to the picogram/mL range by the use of simple chromatographic enrichment techniques (Ackermann and Berna, 2007; Stahl-Zeng et al., 2007). Dozens of targets can be analysed simultaneously:
scheduled MRM, in which selected transitions are coupled to chromatographic windows (Stahl-Zeng et al., 2007), has facilitated analysis of more than 1000 precursor-product ion pairs (Martin et al., 2008). Absolute quantitation can be achieved using appropriate isotope labelled synthetic standards generated either by peptide synthesis (Stemmann et al., 2001) or by recombinant expression of articial genes encoding a concatenation of
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(A)
Discovery Based Approach Fecal Extract
(B)
Hypothesis Based Approach Fecal Extract
Pre-fractionation SDS PAGE

70 fractions
Pre-fractionation SEC
18 fractions
RP HPLC
13 fractions
SDS PAGE
Protein identication using mass spectrometry
Selection of CCAP from literature And excising from gel based on l l weight i ht molecular Identication of protein using MRM
Relative/absolute quantitation using MRM
Fecal MSMS library Relative/absolute quantitation using MRM

Figure 5. Alternative workows for faecal biomarker discovery. (A) Discovery-based approachfollowing alternative sample pre-fractionation strategies (SDS-PAGE, RP HPLC and SEC), which each revealed a number of unique proteins, a faecal MSMS library was developed containing peptide fragmentation data that can be used to generate an MRM assay for large scale quantitative screening of clinical samples (Ang et al., 2009; manuscript in preparation). (B) Hypothesis-based approachproteotypic peptides are selected for potential CRC biomarkers. Samples are pre-fractionated on 1D SDS-PAGE and proteins specically targeted based on their anticipated molecular weight and migration distance. The selected proteins are excised and their presence/absence conrmed using MRM. Relative or absolute quantitation can be carried out across CRC/Normal samples using corresponding regions of the gel (Ang and Nice, 2010).
appropriate tryptic peptides (Pratt et al., 2006). Recently, the availability of low-cost libraries of crude, unpuried synthetic peptides (JPT Peptide Technologies) as reference for validation of peptide identity have enabled high-throughput optimization, validation and development of MRM assays (Picotti et al., 2009a). Examples of the successful use of MRM for the detection of serum proteins include analysis of PSA (Barnidge et al., 2004; Keshishian et al., 2007), C-reactive protein (Kuhn et al., 2004), the multiplexed analysis of 53 medium abundance proteins including L-selectin in a single run (Anderson and Hunter, 2006) and quantitative analysis of a number of N-glycosites including megakaryocyte stimulating factor (Stahl-Zeng et al., 2007). A further advantage of proteomics/MRM analysis is the potential to identify and quantitate protein isoforms (Jenkins et al., 2006). Such isoforms may be linked to abnormal expression at tumour sites (Bourdon, 2007). However all isoforms may not be readily detectable using currently available antibodies (Bourdon, 2007; Lehmann et al., 2008). Proteomics/MRM-based analysis can circumvent this problem by targeting specic peptide sequences
of the dierent isoforms: a recent study (Lehmann et al., 2008) demonstrated the feasibility of simultaneously measuring four isoforms of the sucrose phosphate synthase enzyme that could not be dierentiated by antibodies.
Discovery-based Approach Combining Protein Proling and MRM

The availability of quantitative MRM assays which are specic, sensitive, quantitative and rapid to develop is provoking a major paradigm shift in how biomarker studies are progressed. Key to the discovery phase is the establishment of a comprehensive library of the proteins, proteotypic peptides and associated MS/MS fragmentation data associated with the target sample (Fig. 5A). These data can then be used to develop MRM transitions to conrm their potential for detecting biomarkers of interest in a small set of clinical samples. The validation phase rather than the discovery phase will reveal whether the selected proteins are
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The discovery and validation of colorectal cancer biomarkers viable biomarkers with adequate sensitivity and selectivity either individually or as a panel (Zolg and Langen, 2004). The rst step in the discovery based biomarker approach is therefore to obtain a library of proteins identied in the biological sample of interest. Data generated in-house, obtained from the literature or derived in silico can be used to generate this library. The most reliable data will clearly come from analyses performed on the biological extract under study (in our case faecal samples) since they will be subjected to the same sample workup (sample preparation, chromatographic separation etc.) and most importantly the eect of ion suppression due to the presence of highlevel contaminant proteins, which will be dierent for dierent biological matrixes, will be accounted for. For the development of MRM-based assays, the identity of appropriate peptides and their characteristic peptide fragments is required before they can be programmed for specic targetting on the QQQ mass spectrometer. These MS/MS fragmentation data together with their precursor peptide mass are then used to set up and validate MRM transitions (using synthetic peptides) which can be trialled on a small set of additional clinical samples |(with relative quantitation) to conrm their ecacy for detecting multiple biomarkers of interest prior to investing in expensive assay standards for their absolute quantitation. protein prole as the mice mature and display the disease phenotype thus provide a good starting point for future targeted proteomic/immunological approaches for the detection/ quantitation of proteins involved in the initiation and progression of CRC. The work ow began with sample collection whereby individual murine stools were recovered manually using forceps (the animal will typically defecate when picked up), which avoids contamination of the faecal sample by urine or bedding materials The samples were stored at -70C until use. Following manual disruption of the faeces in 0.15%TFA, aliquots were taken for 1D SDS-PAGE. The gels were then Coomassie stained and sectioned into 1 mm strips which were reduced, alkylated and digested with trypsin prior to protein identication using nanoHPLC coupled to an ion trap mass spectrometry. In total 336 proteins were identied with 115 being of murine origin, 201 from gut bacteria and 18 food related (Ang et al., 2009). Although not a strict comparative experiment, murine homologues of colorectal cancer-associated proteins (CCAP) such as haemoglobin, haptoglobin, haemopexin, alpha-2 macroglobulin and cadherin-17 were identied in the faeces of aged C57BL/ApcMin+ mice, demonstrating the potential of faecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS-based biomarker studies on similar human samples.
The Use of Faecal Proteomics for the Discovery and Validation of CRC Biomarkers
Our hypothesis for developing faecal proteomics was that samples from patients with asymptomatic advanced adenomas or colorectal cancer would contain signature proteins and/or peptides relating to the presence of the adenoma or tumour that are not present in stools from healthy people. These proteins/ peptides should be relatively more abundant in faeces than in blood, thereby facilitating their detection (Kim et al., 2003). Stools therefore prove an ideal non-invasive biological sample for the identication of potential CRC biomarkers. Once potential biomarkers have been identied, MRM can be used for the validation of potential biomarker panels for the detection and monitoring of adenomas and CRC, staging of the disease, response to treatment or recurrance. It is now generally accepted that individual biomarkers are unlikely to have the required sensitivity and specicity and that panels of biomarkers will be required for accurate diagnosis (Duy et al., 2003; Leach et al., 2005; MacBeath, 2002).
Proteins from the Murine Gut Microbiota

The presence of the bacterial proteins in the faecal samples warrants further discussion. Around 60% of the faecal mass in humans consists of bacteria (Guarner and Malagelada, 2003). These bacteria arise from 300500 dierent bacterial species which are predominantly present in the large intestine (Guarner and Malagelada, 2003). Around 40 species account for 99% of the total population (Breitbart et al., 2003) with members of the Bacteroidetes and Firmicutes (including Clostridia) phyla being the most prominent (Eckburg et al., 2005). It is therefore not surprising that in our analysis of the proteins present in the murine stool samples, around 60% were from the microbiota (Ang et al., 2009). Analysis of stools from individual animals showed signicant diversity and relative abundance, as has previously been described for human faeces (Guarner and Malagelada, 2003). Importantly, however, the presence of the bacterial proteins did not appear to interfere with our analysis of the murine faecal proteome. Many of the intact bacteria will be removed during the initial centrifugation steps (13,000g; Cappuccino and Sherman, 2007). Analysis of the MS data suggested that the majority of peptides were of murine origin, as evidenced by the high sequence coverage of these proteins compared with those from the bacteria (Ang et al., 2009).
Murine Faecal Proteomics

The rst study on the murine faecal proteome was undertaken by Oleksiewicz et al. (2005), who used 2D PAGE methodology to identify 28 of the most abundant proteins in murine faeces. Using a mouse model of CRC, we have employed a 1D-SDS PAGE nanoLC-MS/MS based approach to prole the proteome of faeces from C57BL/ApcMin+ mice and the wildtype C57BL mice control (Ang et al., 2009). C57BL/ApcMin+ are highly susceptible to spontaneous intestinal adenoma and polyp formation, with older mice often showing visible signs of rectal bleeding. Typically adenomas are detectable when the C57BL/ApcMin+ mice reach 34 months of age (Moser et al., 1990; Su et al., 1992). We therefore used this model system of colorectal cancer, using individual stools from young (4 W), aged (24 W) and control mice, to investigate the potential of faecal proteomics to identify tumourrelated proteins and peptides in stool samples. The dierences in
Human Faecal Proteomics

Colorectal cancer-associated proteins present in faecal samples could be due to the direct shedding of tumour proteins into the intestinal lumen, inammation reactions or contribution from shed colonocytes (Nair et al., 2003). These could be further broken down into inammatory cells, stromal markers/ inammatory cells, leakage markers/inammatory process, cell shedding, brush border enzymes, angiogenesis or binding proteins. To identify proteins in faeces, stool samples were obtained from either colorectal cancer patients, following diagnosis at
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CS. Ang et al. colonoscopy and prior to surgical resection, or from volunteers who had recently undergone colonoscopy and had no evidence of disease. Samples were collected under the appropriate human ethics approval using a simple standard operating procedure (SOP). Patients or volunteers were given a sample collection kit when they had consented. They were instructed to empty their bladder, and then ush the toilet prior to sample collection. A cellulose biodegradable sample collection sheet was then placed in the toilet bowl to retain the stool sample. Once the stool had been collected, an aliquot was recovered using a plastic spatula and placed into a sample collection tube which was dispatched immediately to the laboratory. Samples were only used for subsequent analysis if received within 2 h of defecation. They were stored immediately at -70C prior to proteomic analysis. Samples (typically 50100 mg) were homogenized in 0.15% TFA, using the protocol we had developed for the murine faecal proteomics (Ang and Nice, 2010) and then processed using the protocol shown in Fig. 5(A). acterized: no human proteins were identied. It was proposed that this was due to low similarity to proteins in the available databases (Klaassens et al., 2007). By contrast, in a recent study using shotgun proteomics of human faecal samples obtained from 37-year-old female healthy monozygotic twins, 2214 nonredundant proteins were identied (Verberkmoes et al., 2009). Although in this study the sample preparation (dierential centrifugation) was specically designed for the recovery of bacterial proteins, almost 30% of the proteins identied were of human origin due to contamination of the bacterial fraction with highly abundant human proteins in the samples with a large number of spectral counts (Verberkmoes et al., 2009).
CCAP Identied in Human Faecal Samples

We have now analysed samples from nine CRC and nine normal subjects and generated a library containing data for more than 350 proteins. This has enabled us to identify more than 2500 proteotypic peptides and their associated MS/MS fragmentation data. These data were acquired using both a LCQ Deca and LTQ Orbitrap and the acquired MS/MS spectra were searched against the LudwigNR database (Eddes et al., 2002; approximately 5,000,000 entries at the time of search) using the MASCOT search algorithm (version 2.1.04, Matrix Science, UK). Searches were conducted with carbamidomethylation of cysteine as a xed modication (+57 Da) as well as variable modications consisting of protein N-terminal acetylation (+42 Da) and oxidation of methionine (+16 Da), with allowance for up to two missed tryptic cleavages. The matched peptides were evaluated using the following criteria: (i) a protein is considered positively identied when there are at least two unique peptides have been identied, each with a MASCOT peptide ion score of at least 35; and (ii) for proteins identied with a single unique peptide, this peptide must have been identied more than once (e.g. in different SDS-PAGE fractions or dierent extraction procedures). Alternatively where manual verication of the spectra was performed, the MS/MS spectrum were required to show good signal-to-noise with prominent peaks well above baseline, exhibits a contiguous series of at least threeb-ory-type ions with the intense ions being accounted for and Mascot peptide ion score well separated from the next highest score. Table 2 shows a small subset of the human proteins identied in this study. Many of the proteins have been previously reported as potential CCAP including CEA6, haemoglobin, complement C3, S100A8, 9 and 12 (Johne et al., 2001; Polanski and Anderson, 2006; Stulik et al., 2000; Ward et al., 2006). Interestingly these include proteins which are present in blood at low levels, such CEA (Kim et al., 2003) and MMP9 (Polanski, 2006), and proteins which have been reported as potential markers of early stage adenoma, such as MMP9 (Gimeno-Garcia et al., 2006; Nielsen et al., 1996).
Multidimensional/Orthogonal Sample Preparation

To investigate the use of multidimensional/orthogonal chromatographic sample preparation to mine deeper into the faecal proteome and reveal low-level components, we took equivalent aliquots of a faecal sample from a male patient who had been diagnosed with colorectal cancer at colonoscopy and analysed them using alternative prefractionation techniques: 1D SDSPAGE, reversed phase HPLC (RP-HPLC) and size exclusion chromatography (SEC) (Fig. 5A). As has been reported previously (Assiddiq et al., 2008; Nice et al., 2007; Tang et al., 2008), dierent subsets of proteins were revealed using alternative separation techniques. In this study, 92 proteins were identied by SDSPAGE (83 of human origin and nine related to bacteria), 41 by RP-HPLC (38 human proteins, two proteins of bacterial origin and one food-related) and 26 by SEC (25 human and one bacterial). Of the human proteins, 15 were common to all three data sets, 28 between the SDS-PAGE and RP-HPLC, 22 between SDS-PAGE and SEC and 16 between SEC and RP-HPLC (Fig. 5A).
Bacterial Proteins Present in Human Faecal Samples

As we had observed with the murine samples, the presence of bacterial proteins in the faecal samples did not appear to interfere with the analysis of human CCAP. Again, signicant dierence was observed both in the number and nature of the bacterial proteins indentied in samples from dierent people. The number of bacterial proteins identied in the stools of CRC patients tended to be lower, perhaps reecting the eects of the bowel preparation which the patients had recently undertaken. However, it was interesting to note that several of the bacteria identied were suggestive of previous or existing pathologies [e.g. Bordetella pertussis (whooping cough), Mycoplasma pneumonia (pneumonia) and Porphyromonas gingivalis (periodontal disease)]. Proteomics analysis of the faecal bacterial proteome could therefore also be of interest. Indeed, in an initial metaproteomics study investigating human infant faecal samples (Klaassens et al., 2007), where soluble protein was extracted and analysed by 2D-PAGE and MALDI-TOF, although more than 200 gel spots were resolved and 50 spots taken for analysis, only two microbial proteins were char-
Faecal Proteomics: From Discovery to Translation

Having identied a number of CCAP, a rapid static MRM assay was established on an Agilent 6410B QQQ using a proteotypic peptide ion and four transitions per peptide. Mass screening was initially performed on a pooled unfractionated faecal extract (extracted directly from the faeces using 0.15% TFA) from eight CRC patients and seven normal volunteers to conrm their routine presence and conrm appropriate high signal intensities on the mass spectrometry. Analyzing unfractionated materials
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Table 2. Subset of CCAP identied in human faeces Function Inamation Stromal markers/inammation Leakage/inammation Cell shedding Brush border enzymes Angiogenesis Protein binding Proteins identied Defensin 1, Defensin 3, Lipocalin, IgGFcBP S100A8, S100A9, S100A12, S100P Haemoglobin, haptoglobin, a1-antitrypsin, lactoferrin A33, CEA, M2PK, MMP9, TIMP1, cadherin-17, DPP1, DPPIV, maltose glucoamylase, CA1, CA2 IGFBP2 Selenium BP, galectin 3BP
Figure 6. Identication and validation of peptides using MRM. (A) Extracted ion chromatogram (EIC) of the an endogenous proteotypic peptide VGAHAGEYGAEALLER (haemoglobin alpha) from the faecal extract of a CRC patient. (B) EIC of the corresponding synthetic peptide VGAHAGEYGAEALLER showing equivalent HPLC retention time and MSMS fragmentation pattern. (C) The y-series transition ions used for the MRM assay. (D) Using the most intense fragment ion (y4), screening of eight CRC patients and seven normal volunteers was carried out. No signal was observed in any of the normal samples.
would help overcome potential problems of the resulting protein fragments having dierent physiochemical properties (e.g dierent molecular weights, pI, hydrophobicity) resulting in assay bias, which is not desirable in a clinical assay. Once good signal-tonoise had been established with the absence of any interfering signals, the corresponding synthetic peptides were then purchased (JBT Peptide Technologies) to conrm the peptide identities. The combination of equivalent chromatographic retention time, peptide mass, fragment mass and fragmentation pattern
for the endogenous and synthetic peptides (e.g. Fig. 6A,B) eliminates ambiguities in peptide identication (Ang and Nice, 2010; Gupta et al., 2009). Optimizations of the peptide collision energies were then carried out using PeptideOptimizer (Agilent). As mentioned above, to be useful as a clinical diagnostic assay, sample manipulation should be kept to a minimum in order to maintain the samples integrity, reduce losses and assay variability and keep costs to a minimum. Sample manipulation steps such as immuno-depletion or prefractionation are generally not
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CS. Ang et al. encouraged as they add a level of bias. We have employed an approach whereby screening was carried out on the unfractionated samples through direct nanoLC-MRM following sample extraction, digestion and sample cleanup to minimize the amount of manipulation following sample collection, similar to that of a clinical laboratory setting. Following screening of individual samples from the eight CRC patients and seven normal volunteers, we have currently identied nine proteins that are only found in the faeces of CRC patients and are in the process of validating the eectiveness of these nine proteins for CRC detection in a larger sample size (manuscript submitted). One of the proteins only identied in the unfractionated faeces from CRC patients is alpha-1 antitrypsin. Alpha-1 antitrypsin, a protein used as a measure of protein leakage into the intestinal track, has been proposed as a good alternative blood-borne marker for CRC since an increase in enteric protein loss from colorectal cancers may also relate to increased epithelial turnover and leakage (Moran et al., 1993; Waldmann, 1985; Ward et al., 2006). Haemoglobin, which forms the basis of the FOBT test, was only found in the faeces of the CRC patients tested (Fig. 6). Haemoglobin was identied from eight transition ions and was further veried using an equivalent synthetic peptide which displayed equivalent HPLC retention time and MSMS fragmentation patterns. Isotopically labelled peptides for absolute quantitation [labelled at the C-terminus with the heavy stable isotope lysine or arginine (13C15N)] were purchased from JPT Peptide Technologies in a 96-well plate format. Once cleaved from the plate with trypsin they were puried by micropreparative HPLC and quantied by amino acid analysis. Known amounts of the labelled peptides were spiked into faecal extracts from eight CRC patients and seven normal volunteers (manuscript submitted). Quantitation using MRM revealed levels of haemoglobin ranges from 6 to 13470 ng peptides/mg stool (2864500 ng protein/mg stool) in CRC patients. There was no detectable level in the normal samples. These data are consistent with the clinical pathologies. denced by their detection in lower molecular weight regions of the gel. Karl et al. (2008) in their study on faecal biomarkers for CRC diagnosis had also shown stability of a number of faecal proteins including CEA, calprotectin and TIMP-1 (Karl et al., 2008). CCAPs such as protocadherin 24, carcinoembryonic antigen 6, S100 calcium binding proteins (S100A6, A8 and A9), prolin-1, L-plastin and selenium binding protein were detected. Identied proteins were validated with their respective synthetic peptides based on equivalent HPLC retention time and MSMS fragmentation patterns (Fig. 6). A relative comparison of the 16 stable proteins was carried out using samples from ve CRC patients and ve normal volunteers. These studies showed that in this sample set haemoglobin, myeloperoxidase, S100A9, lamin A and L-plastin were only present in the faeces of CRC patients. Data for haemoblobin is showed in Fig. 6. Validation of this panel of ve proteins in a larger cohort of patients/normal volunteers will reveal the diagnostic nature of these proteins. This directed process could easily be applied to other biological uids such as serum, urine or semen,or other disease states. This hypothesis-driven approach could also be adapted for other biological studies including pathway analysis, quantication of immuno-anity-enriched proteins, degredomics or analysis of protein complexes. This approach should signicantly reduce the time taken for clinical assay development, with the added advantage that many proteins targeted will have been validated previously using in vitro and/or in vivo studies.
Conclusions
The early years of clinical proteomics saw much discussion as to the success and cost ecacy of such studies (Huber, 2003; Zolg and Langen, 2004). In the eld of biomarker research it was noted that, in spite of the large amount of eort being put into such studies, few proposed biomarkers were achieving FDA approval (Carr and Anderson, 2008; Ludwig and Weinstein, 2005) and that there was a long and uncertain path to clinical utility (Rifai et al., 2006), due largely to problems with clinical validation (Carr and Anderson, 2008). As we enter a new decade it is interesting to see a renewed optimism emerging (Abu-Farha et al., 2009; Pan et al., 2009; Parker et al., 2010). This is being driven largely by the development of novel, sensitive, cost-eective, quantitative MS-based techniques (e.g. MRM; Abu-Farha et al., 2009). These techniques provide a generic platform technology that can be used to address numerous disease situations without the requirement of generating specic reagents. Once MRM assays have been established, they readily lend themselves to multiplex analysis (Picotti et al., 2009b; Schiess et al., 2009). By multiplexing tens to hundreds of potential biomarkers and comparing them across a large number of clinical samples, it will be possible to rapidly screen and assess their suitability. The generation of comprehensive MRM libraries for dierent biological matrices will facilitate a directed, hypothesis driven approach (Ang and Nice, 2010; Schiess et al., 2009; Yang and Lazar, 2009). Importantly, these techniques appear to be highly reproducible within, and across, laboratories (Addona et al., 2009).
Hypothesis-based Biomarker Detection

A second approach to biomarker validation is a hypothesis-based approach whereby selected proteins potentially present in faecal samples, and previously implicated as colorectal cancer associated proteins (CCAP) from published literature, are directly targeted for excision, identication and relative quantication using MRM, based on their predicted molecular weight on SDS-PAGE (Ang and Nice, 2010; Fig. 5B). Such proteins can be selected from previous immunohistochemistry/proteomics studies comparing biological samples such as tumour and adjacent normal tissues, blood plasmas, cell line secretomes and faeces. A caveat to this approach is that proteins may, of course, not be present at detectable levels in the samples analysed, or may not run at their anticipated molecular weight due to either post-translational modication or sample stability. To show proof of principal for this approach we selected a list of 60 proteins reported in literature. Proteins were selected if the met any of the following criteria: (1) prior identication in faeces; (2) if they had been reported to be elevated in the serum of CRC patients; (3) presence in colon and intestinal tissues and cell line; and (4) had been proposed as potential cancer biomarkers in the last 10 years. We were able to directly identify a total of 19 of these proteins in a faecal sample from a patient with colorectal cancer based on their migration on 1D SDS-PAGE, Interestingly only three proteins showed evidence of degradation as evi-
Acknowledgements
C.S.A. and E.C.N. were supported, in part, by research grant 433620 from the Cancer Council of Victoria and a grant from the Department of Innovation, Industry and Regional Development, Australia.
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Special Issue: Review Article

(wileyonlinelibrary.com) DOI 10.1002/bmc.1528

The discovery and validation of colorectal cancer biomarkers

Current Screening Methods

Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

Biological Samples for CRC Biomarker Discovery

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.

View this article online at wileyonlinelibrary.com

CS. Ang et al.

Faecal Protein Biomarkers for CRC Screening

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Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

Proteomics in Colorectal Cancer Biomarker Discovery

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.

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Comparative Proteomics Strategies for the Discovery of CRC Biomarkers

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Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

Cell Line Proling (Secretome Analysis)

Quantitative Biomarker Measurement

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.

View this article online at wileyonlinelibrary.com

CS. Ang et al.

View this article online at wileyonlinelibrary.com

Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

The discovery and validation of colorectal cancer biomarkers

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.

View this article online at wileyonlinelibrary.com

CS. Ang et al.

Discovery Based Approach Fecal Extract

Hypothesis Based Approach Fecal Extract

Pre-fractionation SDS PAGE

Protein identication using mass spectrometry

Relative/absolute quantitation using MRM

Fecal MSMS library Relative/absolute quantitation using MRM

Discovery-based Approach Combining Protein Proling and MRM

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Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

Proteins from the Murine Gut Microbiota

Murine Faecal Proteomics

Human Faecal Proteomics

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.

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CCAP Identied in Human Faecal Samples

Multidimensional/Orthogonal Sample Preparation

Bacterial Proteins Present in Human Faecal Samples

Faecal Proteomics: From Discovery to Translation

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Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 8299

The discovery and validation of colorectal cancer biomarkers

Biomed. Chromatogr. 2011; 25: 8299

Copyright 2010 John Wiley & Sons, Ltd.