**1. Introduction**

332 Biomarker

Tang Y, Forsyth CB, Farhadi A, Rangan J, Jakate S, Shaikh M, Banan A, Fields JZ,

Alcohol-Induced Gut Leakiness and Liver Damage. Alcohol Clin Exp Res.

Keshavarzian A (2009c) Nitric Oxide-Mediated Intestinal Injury Is Required for

Carcinogenesis remains a complex and unpredictable process that involves defects in multiple signalling pathways. Environmental determinants and lifestyle practices may contribute toward their onset by the exposure to a variety of carcinogenic agents. Since the process of carcinogenesis involves the **synergistic induction** in multiple pathways inside the cell, an effective means to investigate and understand them is to engage a global approach that identifies and considers multiple changes simultaneously at the protein level (Albini & Sporn, 2007; Alderton, 2007; Hanahan & Weinberg, 2011; Mueller & Fusenig, 2004). Such an approach can be effectively engaged with the use of discovery proteomics that allows for the large-scale analysis of protein identity and expression (Anderson, Anderson, et al., 2009; Cox & Mann, 2011; Cravatt, Simon, & Yates, 2007; Diamandis, 2004; Nilsson et al., 2010; Walther & Mann, 2010; Wright, Han, & Aebersold, 2005). There is increasing strong evidence that tumorigenesis occurs in the **tissue microenvironment** as a whole, involving the active crosstalk between epithelial, endothelial, immune and stromal cells (Albini &Sporn, 2007; Alderton, 2007; Mueller & Fusenig, 2004). Consequently, the analysis at the whole tissue level is a logical initial step in the identification of tissue-specific or tissueprevalent proteins occurring at larger concentration levels relative to those found in the systemic circulation, wherein their secretion and shedding may occur (Hanash, Pitteri, & Faca, 2008). Provided that the expressed tissue specific and prevalent proteins found in the serum or plasma represent phenotypic cancer pathophysiological events, then these proteins may be potential cancer biomarkers and/or physiologic treatment targets (Hanash, Pitteri, & Faca, 2008).

Research involving the mass spectrometry (MS) based proteomic study of fresh-frozen whole prostate tissue biopsies, cell-culture models and blood sera originating from welldefined clinical designs are discussed. Emphasis is given to those approaches involving the hyphenation of liquid chromatography with mass spectrometry by means of electrospray

<sup>\*</sup> Corresponding Authors

The Discovery of Cancer Tissue Specific Proteins in Serum: Case Studies on Prostate Cancer 335

such as serum and plasma, is poor relative to the natural abundance levels of the tissuespecific secreted or shedded molecular entities of disease, and (iv) the majority of the available analytical protocols measure biomarkers at the DNA and mRNA level, which may not reflect the phenotypic aspects of disease (Adewale et al., 2008; Buchen, 2011; Lin et al., 2005; Rahbar et al., 2011; Sawyers, 2008; Turteltaub et al., 2011). In addition, the availability of more selective prognosis strategies may also help identify patient cohorts, or even single individuals, eligible for adjuvant therapy (i.e., **personalized medicine**). Hence, new biomarkers for asymptomatic prediction, diagnosis, prognosis and response to treatment at the protein level are warranted to improve clinical intervention. It is assumed that one of the critical parameters for the staging of disease and/or treatment intervention is the difference in concentration levels found for the respective biomarkers. This especially becomes true when the complexity of the derived proteomes is decoded in the form of biological pathways and their networks that allow the interrogation of novel candidate protein markers as physiologic targets. Consequent to with this notion, the family of protein markers that will encompass the molecular biology of carcinogenesis will include not only tissue specific proteins but also proteins that reflect systemic changes that predispose a seemingly healthy individual to a longer-term initiation to event of carcinogenesis (Adewale et al., 2008; Buchen, 2011; DeMarzo et al., 2004; Hanahan & Weinberg, 2011; Joyce, 2005; Rahbar et al., 2011; Sawyers, 2008; Turteltaub et al., 2011). In addition to protein markers, and in particular enzyme species, other biological indicators of disease and its predisposition, may include co-factors (i.e., vitamin species) and protein end-products such as 1° and 2° metabolites in the form nucleic acids, amino acids, fatty acids and xenobiotic species in their parent and biotransformed moieties. This integrated monitoring of these biomolecular entities at multiple levels may impart more accuracy and reliability in functionally capturing biochemical pathways of disease. A general example may include an *in vivo* phosphorylation at the catalytic domain of a protein substrate leading to the inhibition of the metabolism of its affiliated ligand. The absence of biotransformed ligand constitutes a proof-positive indicator in the functional annotation of the protein under consideration. A case in point is the polymorphism of the enzyme species 5-methyltetrahydrofolate reductase (5-MTHFR) leading to altered concentration levels of 5-methyl tetrahydrofolate (5MTHF), a metabolically active form of folic acid. The polymorphism of 5- MTHFR has been implicated as a cause to the sub-clinical deficiency of folic acid observed in the older human adult populations, despite their adequate intake of this essential nutrient. The ability, therefore, to quantitatively monitor both the polymorphic 5-MTHFR enzyme and its biotransformation product 5-MTHF can better capture this event (Antoniades et al., 2009; S. D. Garbis, Melse-Boonstra, West, & van Breemen, 2001; Melse-Boonstra et al., 2006; Yetley et al., 2011). The unique analytical versatility and adaptability of MS based methods in detecting diverse biomolecular species imparts a unique opportunity in both customizing and validating key mechanisms of disease and its etiology. From this perspective, modulating these **mechanism based biomarkers** may cause the induction or inhibition of a given carcinogenesis pathway (Kocher & Superti-Furga, 2007). Consequently, such types of biomarkers make for better candidates as treatment targets that can be modulated with medicinal agents and other clinical intervention schemes. Our working hypothesis is based on the assumption that the key difference between the early, asymptomatic disease (low disease burden) versus that of late stage, metastatic disease (high disease burden) is the concentration level found for these mechanistic biomarkers either in their native

ionization (LC-MS) for the analysis of proteins derived from prostate cancer clinical specimens (S. D. Garbis et al., 2011; S. D. Garbis et al., 2008). One of the several challenges of the serum and plasma proteomic methods involve the removal of high abundant proteins (i.e. albumin, IgGs, etc.) for the in-depth analysis of the lower abundant proteins where potential biomarkers can be revealed (S. D. Garbis et al., 2011; Hanash et al., 2008). However, their removal typically results in the co-removal of a significant percentage of the lower abundant tissue specific proteins. At the same token, the co-analysis of both high and low abundance proteins and their endogenously occurring cleavage products (**serum degradome**) may confer greater insight on serum biochemistry and cancer biology. The principle themes to be covered in the present book chapter includes the development and application of quantitative bottom-up LC-MS proteomic methods in the analytical characterization of (i) fresh frozen cancerous breast and prostate tissue biopsy specimens to define proteins expressed by the tumour microenvironment, (ii) the discovery of tissue specific serum biomarkers that are secreted in the systemic circulation of clinical utility to the medical practitioner, (iii) the future perspective on the use of targeted and highthroughput LC-MS based analysis approaches for the validation of biomarker discovery findings spanning large scale specimens sets including healthy specimen cohorts], and (iv) the use of lab-on-chip formats to further enhance LC-MS analysis sensitivity, selectivity and specificity at multiple orders of magnitude lower clinical specimen amounts currently used. The analytical attributes intrinsic to these methods allow the generation of a panel of protein biomarkers with multiple molecular features as reflected on measurable analytical variables that include the chromatographic retention times indexes, the concentration level, the amino acid sequence of the proteolytic peptide, uniquely traceable or surrogate, to one particular protein, and its *in vivo* modification status. The uniqueness in molecular features encoded in a given biomaker panel is accomplished by an ensemble of analytical variables that are explicitly dependent on the collective physico-chemical properties of the proteins and their surrogate peptides that constitute this panel. The end-product from the use of such methods is the determination of tumor "signatures" at the serum or plasma level based on rationally derived protein-panels with a high degree of specificity and sensitivity that uniquely identify a particular cancer type, its stage and its applicability to personalized intervention protocols.
