TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach

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 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.

Keywords: Labeling Efficiency; NHS Ester Chemistry; Peptides*; Phosphoproteome; Post-Translational Modifications*; Quantification; Stable Isotope Labeling; Tandem Mass Spectrometry; Tandem Mass Tags.

Graphical abstract

Fig. 1.

Peptide titration experiments using the vendor recommended (A–C) and a down-scaled (D–F) TMT labeling protocol. (A) Quantities and concentrations of a mix of TMT10-plex reagents (blue) and peptides (gray) are shown for increasing peptide amounts in labeling volumes recommended by the TMT vendor (pep: peptide). The TMT reaction was quenched using 50 mm Tris, pH 8 (final concentration). (B) PSMs identifying underlabeled and fully labeled peptides are depicted for intralaboratory replicates using the labeling protocol displayed in (A). (C) The number of PSMs assigned to overlabeled, O-acylated peptides, and the distribution of serine, threonine, and tyrosine in these spectra are illustrated for the labeling protocol displayed in (A). (D) Same as (A) but using TMTzero and smaller peptide quantities in decreased volumes (pep: peptide). The TMT reaction was quenched using 0.4% hydroxylamine (final concentration). (D) Same as (B) but for the peptide titration row displayed in (D). (F) Same as (C) but for the peptide titration row depicted in (D).

Fig. 2.

TMT titration experiment using the down-scaled TMT labeling strategy across laboratories. (A) Quantities and concentrations of TMTzero reagent (blue) and peptides (gray) are illustrated for increasing TMT quantities in constant labeling volumes (pep: peptide). The TMT reaction was quenched using 0.4% hydroxylamine (final concentration). (B) PSMs identifying underlabeled and fully labeled peptides are shown for intra- and interlaboratory replicates following the protocol depicted in (A). (C) The number of PSMs assigned to overlabeled, O-acylated peptides and the distribution of serine, threonine, and tyrosine in these spectra are displayed for the workflow shown in (A).

Fig. 3.

Benchmarking the optimized protocol for deep-scale (phospho)proteomic analysis. (A) TMT10-plex experiments were performed using five replicates each of peptides derived from basal and luminal breast cancer PDX models and following the two different labeling protocols displayed here. Quantities and concentrations of TMT10-plex reagents (blue) and peptides (gray) used per channel are shown for the standard (4) and the optimized labeling protocol (; pep: peptide). (B) The table lists the number of total PSMs, PSMs identifying fully and partially labeled peptides, and distinct (phospho)peptides for the whole cell and phosphoproteome analyses following the labeling protocols displayed in (A). (C) Bar charts illustrate proteins (upper panel) and phosphosites (lower panel) that were identified for both or only one of the two labeling workflows depicted in (A). Proteins and phosphorylation sites mapping to the human database are given in brackets. (D) Pearson correlation coefficients are plotted for correlations within TMT10-plex experiments (intraplex) and between TMT10-plex experiments (interplex, i.e. inter-workflow) following the protocols depicted in (A).