Search for Potential Markers for Prostate Cancer Diagnosis, Prognosis and Treatment in Clinical Tissue Specimens Using Amine-Specific Isobaric Tagging (iTRAQ) with Two-Dimensional Liquid Chromatography and Tandem Mass Spectrometry

Abstract

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.

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

“…2. A surrogate peptide sequence and its relative quantification indicating the over expression of prostate-specific membrane antigen (PSMA) in prostate cancer (PCa) vs. benign prostate hyperplasia (BPH) with corresponding immunohistochemical confirmation for these specimen categories that effectively corroborate the quantitative proteomic findings (S. D. Garbis et al, 2008).…”

“…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).…”

“…The development and application of a quantitative proteomic method involving the use of off-line size-exclusion chromatography (SCX) followed by the on-line reverse phase (RP) chromatography hyphenated with high resolution, tandem mass spectrometry (2DLC-MS-MS) in combination with the use of isobaric tags for relative and absolute quantification (iTRAQ TM ) was applied to the analysis of clinical whole tissue biopsies derived from patients with benign prostate hyperplasia (BPH, n=10) and prostate cancer (PCa, n=10)(S. D. Garbis et al, 2008). Key advantages to this approach include the ability to conduct multiplex experiments, whereby up to eight samples can be analyzed concurrently under the same 2DLC-MS conditions, resulting in reduced systematic error and increased electrospray ionization efficiency leading to higher sensitivity; in addition, since protein identification and quantification is based on tandem mass spectrometric (MS-MS) evidence, increased selectivity, specificity and confirmatory power are achieved.…”

“…Emphasis is given to those approaches involving the hyphenation of liquid chromatography with mass spectrometry by means of electrospray 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).…”

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…LC-MS based quantitative methods such as label free quantification (Higgs et al, 2005;Neilson et al, 2011;Wang et al, 2006), spectral counting quantification (Neilson et al, 2011), intensity based quantification (Bondarenko et al, 2002;Christin et al, 2011), and also labeling techniques were developed for off-gel analyses. Labeling methods such as isobaric tag for relative and absolute quantification (iTRAQ) (Aggarwal et al, 2006;Chen et al, 2010;DeSouza et al, 2005;Garbis et al, 2008;Muraoka et al, 2012;Wang et al, 2012), and the recently developed isotope coded protein labeling coupled to immunoprecipitation (ICPL-IP) (Vogt et al, 2013), both relying on the labeling of the primary amino groups and lysine residues, were developed. Isotope coded affinity tag (ICAT) (DeSouza et al, 2005), that labels cysteine residues was also applied for tissue proteomics.…”