TMT labeling for the masses: A robust and cost-efficient, in-solution labeling strategy
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�?g 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.
Sample Processing Protocol
An overview of labeling conditions including quantities, volumes, and concentrations of reactants and solvents are listed in the RawfileAnnotations.csv. in brief, increasing peptide quantities (12.5 to 800 �g) were labeled using the same TMT concentration and quantity (100 or 800 �g) and including, in total, 11 conditions as technical, intra-laboratory duplicates or triplicates. Moreover, 17 samples were labeled in three experiments as unicates applying different TMT (40 to 400 �g) and peptide (40 or 200 �g) quantities and concentrations to explore the impact of these parameters on labeling performance and to examine the adaptability of optimized protocol parameters to lower peptide quantities. To assess inter-laboratory robustness, four labeling experiments, in which the TMT quantity was titrated (50 to 400 �g) against a constant peptide amount (100 �g), were carried out as 7 replicates of which 2 or 3 were performed in three independent laboratories. All experiments for method optimization were analysed as single-shot LC-MS/MS runs. To evaluate the utility of the optimized labeling protocol to highly fractionated samples, a deep-scale (phospho)proteome analysis was performed using using 8x less TMT reagent and comparing the results to the original labeling protocol. Briefly, peptides derived from digests of basal (B) and luminal (L) breast cancer PDX models (WHIM2 and WHIM16) were labeled in 5 replicates, randomized within a TMT10-plex experiment (B-L-B-B-L-B-L-L-B-L) and fractionated using high pH reversed-phase (RP) chromatography. After pooling and phosphopeptide enrichment, 24 whole proteome and 12 phosphoproteome fractions were measured by LC-MS/MS.
Data Processing Protocol
For peptide and TMT titration experiments, peptide identification and quantification were performed using MaxQuant (version 184.108.40.206). Tandem mass spectra were searched against the human reference proteome (UP000005640) and/or the mouse reference proteome (UP000000589) supplemented with common contaminants. Separate searches were conducted to check for under- and over-labeling. For under-labeling evaluation, TMTzero or TMT10 was specified as variable modification on lysine and peptide N-termini. For over-labeling assessment, TMTzero or TMT10 was specified as a fixed label on primary amines within a reporter ion MS2 experiment and, additionally, as a variable modification on either histidine or serine, threonine and tyrosine. Other parameters were left as default. Raw files of fractionated (phospho)proteomes were searched against the human and mouse RefSeq database complemented with common laboratory contaminants using Spectrum Mill suite vB.06.01.202. Briefly, a two-cycle fixed/mix modifications search was conducted using the Full, Lys only option which necessitates lysine to be labeled with TMT but allows under-labeling of peptide N-termini. Carbamidomethylation of cysteines was set as additional fixed modification, and N-terminal protein acetylation, oxidation of methionine (Met-ox), de-amidation of asparagine, and cyclization of peptide N-terminal glutamine and carbamidomethylated cysteine to pyroglutamic acid (pyroGlu) and pyro-carbamidomethyl cysteine were specified as variable modifications. For phosphoproteome analysis, phosphorylation of serine, threonine, and tyrosine were allowed as additional variable modifications, while de-amidation of asparagine was disabled. Trypsin Allow P was specified as the proteolytic enzyme with up to 4 missed cleavage sites allowed.
Zecha J, Satpathy S, Kanashova T, Avanessian SC, Kane MH, Clauser KR, Mertins P, Carr SA, Kuster B. TMT labeling for the masses: A robust and cost-efficient, in-solution labeling approach. Mol Cell Proteomics. 2019 PubMed: 30967486