TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach
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 overlabeling 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 interlaboratory 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. 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 overlabeling 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 interlaboratory 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. In bottom-up proteomics, a variety of strategies can be followed to determine quantitative differences in the abundance of proteins and posttranslational modifications (PTMs) (1Bantscheff M. Lemeer S. Savitski M.M. Kuster B. Quantitative mass spectrometry in proteomics: Critical review update from 2007 to the present.Anal. Bioanalytical Chem. 2012; 404: 939-965Crossref PubMed Scopus (581) Google Scholar). Among those, stable isotope labeling of peptides using isobaric reagents such as tandem mass tags (TMTs) enables multiplexing of up to 11 samples (2Thompson A. Schäfer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Hamon C. Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar). Each of these 11 tags can be used to label primary amines in peptide digests via the reaction with the NHS ester-based reactive group. Subsequently, all samples are pooled and further processed together, thus reducing technical variation in the experimental workflow. Inside the mass spectrometer, the isobaric nature of the tags leads to a summation of each peptide signal from all labeled and combined samples in the MS1 spectrum. Following peptide fragmentation, sample-specific reporter ions of different mass-to-charge (m/z) values are generated from the different tags owing to the different combinations of heavy carbon and nitrogen isotopes present in the reporter ions. This enables the differentiation and relative quantification of peptides from all conditions in the same MS2 scan. Multiplexing in this manner greatly reduces the number of missing peptide quantification values in each TMT experiment. Further, quantification reproducibility is less sensitive to performance variations of the liquid chromatography (LC) 1The abbreviations used are:LCliquid chromatographyMSmass spectrometryNCEnormalized collision energyNHSN-hydroxysuccinimidePDXpatient-derived xenograftPSMpeptide spectrum matchRPreversed phaseTMTtandem mass tags. and mass spectrometry (MS) systems than for label-free measurements. In addition, the multiplexing capability of TMT reagents enables achieving deep proteome coverage for multiple samples in a reasonable amount of measurement time. Together these advantages render isobaric tags attractive for MS-based proteoform studies, including posttranslational modifications analyses that depend on robust quantification of single peptides across conditions (3Zecha J. Meng C. Zolg D.P. Samaras P. Wilhelm M. Kuster B. Peptide level turnover measurements enable the study of proteoform dynamics.Mol. Cell Proteomics. 2018; 17: 974Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The high intra- and interlaboratory reproducibility of a TMT workflow for deep-scale proteome and phosphoproteome analyses has recently been demonstrated for patient-derived xenograft (PDX) models of breast cancer (4Mertins P. Tang L.C. Krug K. Clark D.J. Gritsenko M.A. Chen L. Clauser K.R. Clauss T.R. Shah P. Gillette M.A. Petyuk V.A. Thomas S.N. M. T. workflow for deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid 2018; PubMed Scopus Google and the of TMT labeling for the analysis of cohorts of T. Krug K. A. P. Gillette M.A. Clauser K. M. Tang L.C. J. J. J. P. A. M. and analyses 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). liquid chromatography mass spectrometry collision N-hydroxysuccinimide patient-derived xenograft peptide spectrum tandem mass tags. of quantification by isobaric labeling is the high cost of and protocols in the amount of TMT reagent recommended by the was reduced G. P. S. S. Kuhn K. labeling of complex peptide digests with peptide for PubMed Scopus Google A. quantitative for proteome of cell J. in and B. J. Zolg D.P. Kuster B. for and efficient of proteome and phosphoproteome L. P. and J. tandem mass efficient strategy for quantitative using tandem mass 2018; 17: PubMed Scopus Google protein A and strategy for TMT 2018; 17: PubMed Scopus Google and applied such optimized labeling to a variety of (3Zecha J. Meng C. Zolg D.P. Samaras P. Wilhelm M. Kuster B. Peptide level turnover measurements enable the study of proteoform dynamics.Mol. Cell Proteomics. 2018; 17: 974Abstract Full Text Full Text PDF PubMed Scopus (66) Google Wilhelm M. B. S. M. S. Kuster B. of in cancer PubMed Scopus Google J. M.A. Quantitative mass multiplexing the abundance of S. proteins across 10 carbon Proteomics. PubMed Scopus Google M. L. M. M. in and proteome from PubMed Scopus Google Scholar). However, on the quantities and concentrations of in the to the of of the of reducing TMT-to-peptide ratios on the overall labeling performance has been to In the present we systematically evaluated the impact of labeling reaction parameters and a robust and efficient TMT labeling protocol that achieves complete labeling of primary amines in peptides using eight less TMT reagent than recommended by the vendor. of the protocol between provide guidance on the of the optimized for different peptide and the of the protocol to proteomic and The of the experimental is in in the and a of labeling conditions including and concentrations of and are in the In peptide quantities to 800 were labeled using the same TMT and quantity or 800 and in 11 conditions as or samples were labeled in as different TMT to and peptide or quantities and concentrations to the impact of these parameters on labeling performance and to the of optimized protocol parameters to peptide quantities. interlaboratory labeling in the TMT quantity was to against a peptide amount were as of or were in for were as evaluate the utility of the optimized labeling protocol to a deep-scale analysis was as (4Mertins P. Tang L.C. Krug K. Clark D.J. Gritsenko M.A. Chen L. Clauser K.R. Clauss T.R. Shah P. Gillette M.A. Petyuk V.A. Thomas S.N. M. T. workflow for deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid 2018; PubMed Scopus Google using the optimized protocol using less TMT and to the labeling peptides derived from digests of and breast cancer models and were labeled in a and using high and proteome and phosphoproteome were by and were in and respectively, with and and and breast cancer models were generated as S. J. A. S. J. C. L. C. M. T. J. C. L. C. T. C. C. A. C. J. C. T. M. L. K. S. A. 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