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Molecular & Cellular Proteomics 7:1162-1173, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Discovery and Verification of Head-and-neck Cancer Biomarkers by Differential Protein Expression Analysis Using iTRAQ Labeling, Multidimensional Liquid Chromatography, and Tandem Mass Spectrometry^*,S

Ranju Ralhan^,,¶,||, Leroi V. DeSouza^,, Ajay Matta^¶,**, Satyendra Chandra Tripathi^¶, Shaun Ghanny^,, Siddartha Datta Gupta, Sudhir Bahadur^¶¶ and K. W. Michael Siu^,,||||

From the Departments of Chemistry and Mathematics and Statistics and Centre for Research in Mass Spectrometry, York University, Toronto, Ontario M2J 1P3, Canada and Departments of ^¶ Biochemistry, Pathology, and ^¶¶ Otorhinolaryngology, All India Institute of Medical Sciences, New Delhi 110029, India

Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3); YWHAZ (14-3-3); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin (PTHA); L-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

^|||| To whom correspondence may be addressed: Dept. of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele St., Toronto, Ontario M2J 1P3, Canada. Tel.: 416-650-8021; Fax: 416-736-5936; E-mail: kwmsiu{at}yorku.ca

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