**3. Analytical chemistry background**

The selection of the analytical method for the investigation of a biological specimen depends on the clinical query to be solved. However, the essential features to this selection process is for the analytical method to impart: (a) qualitative effectiveness for the identification of as many proteins as possible, (b) quantitative effectiveness to reveal absolute or relative concentrations of these proteins. The advent of mass spectrometry (MS) based techniques as compared to the other analytical techniques (i.e., molecular spectroscopy based such as fluorescence, UV-VIS, NMR; X-Ray crystallography) has allowed for the simultaneous protein identification and quantification in very small amounts of biological material, at analyte detection limits that may exceed those of fluorescence based ELISA assays as applied to clinical specimens (S. D. Garbis et al., 2011; Rubakhin, Romanova, Nemes, & Sweedler, 2011). A major milestone to the effective use of MS techniques to the analysis of a vastly larger range of biomolecules (e.g., metabolites, peptides, proteins, nucleic acids, fatty acids, steroids, etc.) was the advent of the electrospray ionization source (ESI) and its micro-

Fig. 1. A key parameter to the utility of a given biomarker is its concentration level in the assessment of disease and its treatment. The current detection methods detect biomarker levels that reflect late stage disease wherein treatment options are limited. The more sensitive and selective the analysis method the greater the effectiveness in capturing the disease progression at the initiation stage wherein its reversal is possible with cancer chemoprevention, nutritional/functional food intervention, and other low-toxicity

or *in vivo* modified form (i.e., post translational modified proteins, biotransformed 1° and 2° metabolites). As such, the ability to capture very low levels of these protein markers and their surrogate end-products provides greater assurance in capturing their disease potential at the progression or even initiation stage whose effects can be reversed with less toxic intervention protocols (see Figure 1). The present discourse will focus on protein-based

The selection of the analytical method for the investigation of a biological specimen depends on the clinical query to be solved. However, the essential features to this selection process is for the analytical method to impart: (a) qualitative effectiveness for the identification of as many proteins as possible, (b) quantitative effectiveness to reveal absolute or relative concentrations of these proteins. The advent of mass spectrometry (MS) based techniques as compared to the other analytical techniques (i.e., molecular spectroscopy based such as fluorescence, UV-VIS, NMR; X-Ray crystallography) has allowed for the simultaneous protein identification and quantification in very small amounts of biological material, at analyte detection limits that may exceed those of fluorescence based ELISA assays as applied to clinical specimens (S. D. Garbis et al., 2011; Rubakhin, Romanova, Nemes, & Sweedler, 2011). A major milestone to the effective use of MS techniques to the analysis of a vastly larger range of biomolecules (e.g., metabolites, peptides, proteins, nucleic acids, fatty acids, steroids, etc.) was the advent of the electrospray ionization source (ESI) and its micro-

treatment protocols.

markers of prostate carcinogenesis.

**3. Analytical chemistry background**

and nano- flow derivatives (Wilm, 2011). As a soft-ionization source, ESI made it possible to introduce the thermally labile biomolecular species to become introduced to the gas phase from its initial liquid phase in its charged state with an intact chemical integrity. Consequently, the ESI source allowed the interfacing of liquid phase sample introduction systems (i.e., liquid chromatography and capillary electrophoresis) with the vacuum-system encased MS platforms (i.e., quadrupolar, ion trapping, time-of-flight, or hybrids thereof, etc.). (Cox & Mann, 2011; Cravatt et al., 2007; Diamandis, 2004; Kocher & Superti-Furga, 2007; Nilsson et al., 2010; Walther & Mann, 2010). The development of novel analytical methods that are based on the combined use of liquid chromatography and tandem mass spectrometry (LC-MS) techniques for the bottom-up or top-down proteome analysis of a wide spectrum of both low and high abundant proteins in clinical tissue and sera dates back to the late nineties with the introduction of the Multi-Dimensional Protein Identification Technology (MudPIT) by John Yates (Fournier, Gilmore, Martin-Brown, & Washburn, 2007). The MuDPIT approaches constituted an alternative to the Two-Dimensional Gel Electrophoresis (2DGe) approaches in their ability to capture and identify a wider spectrum of proteins and at lower abundance levels. These in-depth LC-MS proteomic methods employ the orthogonal use of various high-performance liquid chromatographic (HPLC) chemistries, based on the principles of strong ion exchange (XIC), size-exclusion (SEC), hydrophilic interaction (HILIC), affinity capture (biological and chemical), reverse phase (RPC) and others. These separation techniques allow the isolation, separation and enrichment of proteins and surrogate peptides found in extracts derived form clinical specimens such as tissues, blood plasma and sera. Overall, the LC-MS proteomic methods incorporate the combined use of both nano-electrospray ionization (nESI) and off-line matrix-assisted laser desorption ionization (MALDI) interfaces, to ensure the broadest possible surrogate peptide coverage for a given protein. The bottom-up analysis approach, which is based on the analysis of surrogate tryptic peptides, is well suited for a robust and sensitive protein analysis strategy (taking into consideration individual protein hydrophobicity, charge, or post-translational modification). These complementary methodological approaches provide a more comprehensive and reproducible proteomics assessment of clinical tissue and sera specimens. This has become yet more evident with the use of the latest tandem MS-MS analyzer platforms that include the quadrupole time-offlight QqTOF and Orbitrap based geometries. These MS platforms exhibit high-sensitivity (limit of detection < 10 fmol on-column allowing the use of very low signal accumulation times) and ultra-high resolution (≥ 30,000, translating to 1-3 ppm mass accuracies) at very high signal sampling speeds (≥ 30 Hz). Such performance characteristics allow the detection > 3,000 proteins at > 99% confidence derived from cell culture lysates and spanning over 4 orders of magnitude natural concentration abundance in a single LC-MS analysis run (Cox & Mann, 2011; Liu, Belov, Jaitly, Qian, & Smith, 2007; Mann & Kelleher, 2008; Ong & Mann, 2005). One of several key advantages of the non-gel LC-MS based methods is that they allow the analysis of a much wider spectrum of proteins than that typically covered with the classical gel-based approaches. This spectrum includes proteins that are membrane bound or membrane associated; proteins that exhibit alkaline (pI > 8) and acidic (pI < 5) character; proteins with low (<10 kDa) or high (>200 kDa) molecular weights; and proteins that have undergone *in vivo* modifications (i.e. phosphorylation, acetylation, methylation, glycosylation, etc.) occurring in minor molar ratios (oftentimes < 1:1000) relative to their

The Discovery of Cancer Tissue Specific Proteins in Serum: Case Studies on Prostate Cancer 339

**4.1 The quantitative proteomic profiling of clinical whole tissue biopsies derived from** 

Prostate whole tissue biopsies exhibit extensive biological variability when accounting for the diversity in human subjects and the heterogeneity and size of the tissue specimen itself. These variables must be taken into consideration when executing its proteomic study. Factors such as tissue procurement, histopathology pre-assessment, storage, handling, and pre-analytical processing, and instrumental performance verification with standardization (chromatographic and nano-ESI ionization efficiency, MS and MS-MS sensitivity, resolution, accuracy and precision) are variables that need to be optimized for any given proteomic study. The optimization of these variables will minimize the histopathological, biological, pre-analytical and analytical variability so essential to a reproducible and information-rich proteomic output (Buchen, 2011; Cox & Mann, 2011; Diamandis, 2004; Hilario & Kalousis,

Several multiplex proteomics studies that rely on the use of cysteine-specific isotope-coded affinity tags (cICAT), stable isotope labeling with amino acids in cell culture (SILAC), difference gel electrophoresis (DIGE) and trypsin-mediated 18O isotope labeling have been successful in detecting differentially expressed proteins in combined specimen samples (DeSouza et al., 2005; Everley et al., 2004; Hood et al., 2005). Despite their advantages however, intrinsic limitations exist for each of these approaches. The cICAT approach allows only the labeling of proteins containing cysteine residues on tractable peptides upon proteolysis making this approach unsuitable as a comprehensive and in-depth protein discovery tool. The cICAT approach has been used for the quantitative proteomic profiling in secondary prostate cancer cell cultures. In one such study, 524 secreted proteins were from the LNCaP neoplastic prostate epithelium of which 9% of these were found to be differentially expressed (Martin et al., 2004). In another study involving the same cell culture model in response to androgen exposure resulted in the identification of 1064 proteins of

Another label-based approach for prostate biomarker discovery efforts makes use of heavy

phase trypsinization process thus allowing the trypsin-mediated 18O stable isotope incorporation (18O labeling) for those proteins extracted from one specimen category (i.e. control, treated or diseased states). This process leads to the exchange of two equivalents of 16O with two equivalents of the 18O stable isotope at the carboxyl terminus of the resulting tryptic peptides coined as the «heavy» peptides. The heavy water approach was applied to proteins extracted from benign prostate hyperplasia (BPH) vs. prostate cancer (PCa) cells isolated from a single formalin-fixed paraffin embedded (FFPE) prostate cancer tissue specimen (Hood et al., 2005). This study resulted in the quantitative profiling of only 68 proteins. The limited proteins amounts along with their cross-linked form limit the utility of FFPE as a viable specimen source for proteomic assessment. Another confounding factor in the practical utility of the 18O labeling strategy, which also applies in cICAT labeling case, is

A gel-based relative quantitative approach that has been used for prostate cancer cells is known as the differential gel-electrophoresis (DIGE). The DIGE method represents a variant

18O water is used instead of regular water for the solution

which approx. 21% of these proteins were modulated (Wright et al., 2004).

that only two samples can be analyzed per experiment.

**4. Prostate cancer**

2008; Nilsson et al., 2010).

water. In such an approach, H2

**benign prostate hyperplasia and prostate cancer** 

native counterparts (S. Garbis, Lubec, & Fountoulakis, 2005; Lubec & Afjehi-Sadat, 2007; Nilsson et al., 2010; van Bentem, Mentzen, de la Fuente, & Hirt, 2008). Currently more than 150 different types of *in vivo* modifications are possible (Seymour et al., 2006; Shilov et al., 2007). The ability to detect and discriminate these post-translational modified proteins constitutes a major advancement in the more comprehensive understanding of signaling cascades at the protein level allowing for a more direct appreciation of protein-protein interaction and consequently biological pathways and their networks (Kocher & Superti-Furga, 2007; Mann & Kelleher, 2008; Ong & Mann, 2005; van Bentem et al., 2008). It is assumed that the vast majority of proteins have undergone multiple and diverse *in vivo* modifications that define their induction or silencing status. Such protein traits can only be captured with tandem MS spectra generated at high sensitivity and high resolution providing unequivocal evidence in the annotation of their *in vivo* modification at the precise amino acid location in single LC-MS experiment (Liu et al., 2007; Mann & Kelleher, 2008; Ong & Mann, 2005; Papayannopoulos, 1995). Conceptually, a vast array of *in vivo*  modifications can be captured and stored for later use as means to provide a multifactorial understanding of biological pathways and their networks. The current biochemical assays such as Immunohistochemistry and Western blots fail to account for these intrinsic protein *in vivo* modification traits. It is this limitation that has often resulted in the analysis bias between the MS and biochemical assay measurements (Diamandis, 2004; Lubec & Afjehi-Sadat, 2007; Nilsson et al., 2010).

The collective LC-MS analysis characteristics constitute a major advancement toward an in-depth proteome analysis of the fresh-frozen tumor specimens.Advanced proteomics approaches can bridge the gap between the genetic and epigenetic alterations underlying cancer and cellular physiology. The precepts of multidimensional liquid chromatography hyphenated with high resolution, tandem mass spectrometry (MDLC-MS-MS) techniques in combination with the use of isobaric tags for relative and absolute quantification (iTRAQTM) of whole tissue biopsies of various types of cancer tissue (i.e., breast, prostate, cervical) has played a key role in bridging this gap. In general, a key advantage of 2DLC-MS-MS methods that utilize isobaric stable isotope based approaches (i.e., cICAT, TMT, iTRAQ, etc.) is the ability to conduct multiplex experiments, whereby specimen extracts can be analyzed concurrently under the same experimental conditions. This multiplexing advantage reduces systematic error, and improves the signal-to-noise of the precursor MS and product ion MS-MS response allowing for a greater number of proteins to be quantitatively profiled (DeSouza et al., 2005; S. D. Garbis et al., 2008; Glen et al., 2008; Pichler et al., 2011; Wu, Wang, Baek, & Shen, 2006). Advancements made to liquid chromatography and mass spectrometry stand to further potentiate the utility of these isobaric stable isotope tags (Fournier et al., 2007; Pichler et al., 2011). Other key attributes that make MS based methods the premier choice for the analysis of small amounts of clinically valuable and complex biological specimens along with reduced requirements for stable isotope reagents is driven by the increased automation and miniaturization imparted by lab-on-a-chip formats (Everley, Krijgsveld, Zetter, & Gygi, 2004; Koster & Verpoorte, 2007; Rubakhin et al., 2011; Tsougeni et al., 2011). These themes are covered within the context of case studies in the analysis of clinical whole tissue biopsies and their sera for prostate cancer.
