Helmholtz Gemeinschaft


TMT labeling for the masses: a robust and cost-efficient, in-solution labeling approach

Item Type:Article
Title:TMT labeling for the masses: a robust and cost-efficient, in-solution labeling approach
Creators Name:Zecha, J. and Satpathy, S. and Kanashova, T. and Avanessian, S.C. and Kane, M.H. and Clauser, K.R. and Mertins, P. and Carr, S.A. and Kuster, B.
Abstract:Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and over-labeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mM and 2 g/L, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99 % and excellent intra- and inter-laboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
Keywords:Tandem Mass Tags, Stable Isotope Labeling, Labeling Efficiency, NHS Ester Chemistry, Peptides, Phosphoproteome, Post-Translational Modifications, Quantification, Tandem Mass Spectrometry
Source:Molecular & Cellular Proteomics
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:1468-1478
Date:1 July 2019
Official Publication:https://doi.org/10.1074/mcp.TIR119.001385
External Fulltext:View full text on PubMed Central
PubMed:View item in PubMed

Repository Staff Only: item control page

Open Access
MDC Library