Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing

Kevin A Welle,Tian Zhang,Jennifer R Hryhorenko,Shichen Shen,Jun Qu,Sina Ghaemmaghami

doi:10.1074/mcp.m116.063230

Abstract

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

Highlights

Analysis of protein expression is central to our understanding of cellular physiology and regulation
As protein turnover leads to the gradual disappearance of stable isotope labeling in cell culture (SILAC)-unlabeled and appearance of SILAC-labeled MS1 peaks, the kinetics of this process are revealed by the relative intensities of the tandem mass tags (TMT) reporter ions in the MS2 spectra
The relative intensities of TMT reporter ions generated by SILAC-unlabeled precursor peptides monitors their clearance and the relative intensities of TMT reporter ions generated by SILAC-labeled precursor peptides monitors their accumulation

Summary

Introduction

Analysis of protein expression is central to our understanding of cellular physiology and regulation. Dynamic (i.e. time-resolved or pulsed) SILAC experiments (Fig. 1) can effectively measure the labeling kinetics of proteins and provide rates of protein degradation and synthesis on a global scale. For analysis of degradation kinetics in quiescent cells, the ten samples derived from different SILAC labeling time-points were tagged with TMT-126, TMT-127N, TMT-127C, TMT128N, TMT-128C, TMT-129N, TMT-129C, TMT-130N, TMT-130C, and TMT-131 in order of increasing time.

Results

Conclusion