Biomarker Discovery in Low-Grade Breast Cancer Using Isobaric Stable Isotope Tags and Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry (iTRAQ-2DLC-MS/MS) Based Quantitative Proteomic Analysis

Abstract

The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p < or = 0.05). A quantitative comparison revealed 3/3 proteins with significantly increased/decreased level in metastatic primary tumor and 13/6 proteins with increased/decreased level in lymph node metastasis compared to nonmetastatic primary tumor (p < 0.01). Changes in selected differentially expressed proteins were verified with qRT-PCR. Although our pilot scale study does not warrant general biological conclusions, the synergic regulation of some proteins with related function (e.g., heme binding proteins, proteins of energetic metabolism, interferon induced proteins, proteins with adhesive function) determined in our sample set reflects the ability of our method in providing biologically meaningful data. The main conclusion from this pilot study was that our quantitative proteomic method constitutes a novel way of analyzing cancerous breast tissue biopsy samples that can be extended as part of a larger scale biomarker discovery program.

Combined Proteomics and Transcriptomics Identifies Carboxypeptidase B1 and Nuclear Factor κB (NF-κB) Associated Proteins as Putative Biomarkers of Metastasis in Low Grade Breast Cancer

“…Shotgun proteomics with isobaric tags for relative and absolute quantification (iTRAQ) is an established approach for quantification of proteins related to cancer metastasis (6,7). Moreover, recent developments made to multidimensional liquid chromatography and mass spectrometry, including the FT-Orbitrap detector technology, have significantly advanced the discovery proteomics field (8).…”

Differential Protein Expression Profiling by iTRAQ-Two-dimensional LC-MS/MS of Human Bladder Cancer EJ138 Cells Transfected with the Metastasis Suppressor KiSS-1 Gene

“…Mass spectrometry techniques have become the dominant means of protein identification (20). The use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with multidimensional liquid chromatography (LC) and tandem MS analysis (21) is emerging as a powerful methodology in the search for diseasespecific targets and biomarkers using cell lines (22)(23)(24)(25)(26)(27)(28)(29)(30), tissues (31)(32)(33)(34)(35)(36)(37), and body fluids (38,39). The iTRAQ method places tags on primary amines (NH 2 -terminal or -amino group of the lysine side chain), allowing detection of most tryptic peptides.…”

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

“…Comparative/quantitative analysis of highquality clinical biospecimen (e.g., tissue and biofluids) of human cancer proteome landscape can potentially reveal protein/peptide biomarkers responsible for this disease by means of their altered levels of expression, PTMs as well as diff erent forms of protein variants. Despite technological advances in proteomics, major hurdles still exist at every step of the bio marker development pipeline [52][53][54][55][56][57][58][59][60][61][62][63].…”