Study on the reproducibility of TMT coupled with LC-MS/MS technology in analysis of nonfunctional pituitary adenoma proteome

Abstract

Objective To investigate the reproducibility of tandem mass spectrometry tags (TMT) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) technology in analysis of nonfunctional pituitary adenoma proteome and obtain reliable differentially expressed proteins. Methods Tissue sample was subjected to protein extraction, trypsin digestion, TMT labeling, high performance liquid chromatography (HPLC) fraction, LC-MS/MS, database search, and data analysis. Results SDS-PAGE lanes and bands were visualized clearly without smearing, which indicates no degradation of proteins and the prepared protein sample met the experimental requirement. A total of 6076 identified proteins including 4666 quantitative proteins met the requirement of quality control validation of MS data. The reproducible analysis on 3-plex TMT labeling of each sample found that the average coefficient of variation (CV) in both groups was below 20% in 99% of the quantified proteins, and the average fold change of about 100% of the quantified proteins in both groups was less than 1.6, respectively. The Pearson correlation coefficient (r) in both groups was between 0.9~1. These data demonstrated that TMT quantitative proteomics approach was high reproducible in analysis of each sample. Conclusions The TMT coupled with LC-MS/MS technology is an efficient quantitative proteomics approach with high throughput and high reproducibility. Key words: Tandem mass spectrometry; Chromatography, liquid; Pituitary neoplasms/ME; Proteome