Proteomic Analysis of HPV+ Head-and-Neck Cancer by Quantitative Mass Spectrometry: Molecular Biology and Therapeutics

Abstract

Purpose/Objective(s): A central goal in tumor biology is to describe the molecular processes responsible for transformation of a normal cell into a cancerous one. These processes are encoded by the genome (and epigenome) but are manifested at the protein level. The relationship between the genome of any given cell and its proteome is highly complex, reflecting the immense level of transcriptional/posttranscriptional regulation. Here, we used quantitative mass spectrometry to characterize the HPV positive head and neck squamous cell cancer (HNSCC) proteome and provided an initial map describing the protein expression profile of both the normal and cancerous tissues of head and neck cancer patients. The resources to be developed here will provide further insights to the mechanisms of the pathogenesis of HNSCC. Materials/Methods: HNSCC tissue samples were surgically removed and were snap-frozen in liquid nitrogen. Proteins were extracted and were digested using trypsin. Peptides were then labeled with the isobaric Tandem Mass Tagging (TMT) reagents, and fractionated by HPLC, and were analyzed on a Thermo LTQ-Velos-Pro Orbitrap mass spectrometer. The TMT reagents allow for the analysis of a 6-plex mixture of samples (eg, normal and cancerous tissues from 3 patients). After combining, MS analysis results in only one peak for every peptide in an MS1 scan due to the identical but stable-isotope-balanced mass addition in the reagents. However, the abundance of the peptide in the 6 samples is fully decoded in MS2 spectra using the HCD (High energy collision-induced dissociation) technology. Results:We initially characterized the normal/cancerous tissue pair from 6 HPV+ (as indicated by positive p16+ immunostaining) HNSCC patients. For each sample, 100 mg of protein was used. We identified and quantified a total of 5,941 proteins across these specimens, many of which had a differential expression level between the normal and cancerous samples. We performed Gene Ontology analysis, and found the proteins that were overexpressed in the cancerous tissue were enriched for biological processes including antigen processing and presentation (P Z 8.0E-6), cytokine production (P Z 1.4E-4) and defense response (P Z 8.7E-3). Conclusions: We have developed and optimized a multiplexed and quantitative mass spectrometry pipeline for the analysis of the HNSCC proteome. We applied this technology to initially characterize the tissue sample from a small cohort of HPV+ HNSCC patients. Our preliminary results indicate that immune response might be a key factor in the pathogenesis of at least a subset of these patients. Author Disclosure: Z. Wang: None. Y. Zhang: None. P. Vo: None. B.D. Sumer: None. Y. Yu: None.