Article pubs.acs.org/ac
High-Resolution Enabled TMT 8‑plexing Thilo Werner, Isabelle Becher, Gavain Sweetman, Carola Doce, Mikhail M. Savitski,* and Marcus Bantscheff* Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany S Supporting Information *
ABSTRACT: Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (15N, 13C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between 15N- and 13C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, 12C8H1615N1+; TMT127H, 12C713C1H1614N1+; TMT129L, 12C613C2H1615N1+; and TMT129H, 12C513C3H1614N1+). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays.
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TMT reagents have been designed to efficiently fragment between the mass-balancing and reporter portions upon beamtype collision-induced fragmentation in a collision cell, thus generating reporter ions specific for every flavor of the labels. Alternative fragmentation mechanisms have also been successfully applied, such as pulsed-Q dissociation (PQD) of peptides in ion-trap instruments26 or electron transfer dissociation (ETD).27,28 Multiplexed quantification is enabled by distributing stable isotopes of carbon, nitrogen, or oxygen across the mass-balancing and reporter parts of the reagents, such that reporter ions of different mass are generated without altering the total mass of the reagents. Hence, in order to generate isobaric tagging reagents that afford a higher multiplexing rate, larger reagents have been synthesized, offering more possibilities for the incorporation of stable isotopes. Consequently, the 8-plex iTRAQ29 reagent is a much larger molecule than the 4-plex iTRAQ. However, recent reports indicated lower protein and peptide identification rates for larger labels.30 For example, Pichler et al. observed ∼5-fold fewer peptide identifications in LC-MS/MS analyses of trypsindigested HeLa cell extracts when using the 8-plex, compared to the 4-plex iTRAQ reagent. Interestingly, the 6-plex TMT reagent had an intermediate performance, despite its chemically distinct structure. To a large extent, the impaired identification of peptides labeled with larger tags was found to be caused by additional internal fragmentation of the larger tags. Another recent study comparing different isobaric tags for phosphopro-
nitial efforts in mass spectrometry (MS)-based proteomics focused on the qualitative description of the protein complement of cells and organisms. In recent years, a robust quantitative dimension was added to the analysis, based on either label-free or stable isotope labeling (SIL) approaches.1 This enabled the differential and dynamic analysis of proteins and their post-translational modifications in a wide range of applications, including protein expression profiling,2−4 protein−protein interaction studies,5,6 cell signaling analysis,7−9 chemoproteomic target profiling,10−14 and biomarker studies.15−19 Isobaric (or tandem) mass tags20 such as iTRAQ21 and TMT22 are popular peptide and protein labeling reagents in quantitative proteomics. In contrast to other stable isotope labeling-based techniques such as SILAC,23 the different isotope-coded variants of each reagent produce isobarically labeled peptides and, hence, cannot be distinguished in MS1 scans. However, when peptides are fragmented in the mass spectrometer, a specific low-molecular-weight reporter ion is generated for each of the isotope coded variants of the tag and relative quantification can be achieved by comparing the reporter ion signals, which correspond to the respective samples under investigation. Isobaric mass labeling reagents typically consist of three parts: (1) a reactive group that enables selective modification of peptides and proteins, (2) a massbalancing part, and (3) the reporter part. Most labels utilize Nhydroxysuccinimide esters for modification of ε-amino groups of lysine residues and the N-terminal α-amino groups of peptides. Alternatively, reagents selectively targeting sulfhydryl groups of cysteine24 and carbonyl-reactive groups enabling modification of glycans25 have been reported. The iTRAQ and © 2012 American Chemical Society
Received: June 6, 2012 Accepted: July 30, 2012 Published: July 30, 2012 7188
dx.doi.org/10.1021/ac301553x | Anal. Chem. 2012, 84, 7188−7194
Analytical Chemistry
Article
millliliter (1 mL) of a 1/1/1 mixture of HEK293, K562 and placenta extracts (5 mg/mL protein concentration) was incubated with dasatinib or staurosporine for 45 min at 4 °C over a range of concentrations, (0 (DMSO control), 0.00732, 0.0293, 0.11719, 0.46875, 1.875, 7.5, and 30 μM for staurosporine and 0, 0.00064, 0.00386, 0.02315, 0.13889, 0.83333, 5, and 30 μM for dasatinib) followed by incubation with kinobeads for 1 h in an end-over-end mixer at 4 °C. Beads were transferred to disposable columns (MoBiTec), washed with lysis buffer containing 0.2% NP-40, and eluted with 50 μL of a 2× SDS sample buffer. Proteins were alkylated with 200 mg/mL iodoacetamide for 30 min, separated on 4−12% NuPAGE (Invitrogen), and stained with colloidal Coomassie. Gels were cut into slices across the entire separation range and subjected to in-gel digestion. Peptide extracts were labeled with the eight TMT reagents in 40 mM triethylammoniumbicarbonate (TEAB), pH 8.53. After quenching the reaction with glycine, labeled extracts were combined. Extracts from vehicletreated samples were labeled with TMT reagent 131, and combined with extracts from compound-treated samples labeled with TMT reagents 126−130, including the “new” labels. Experiments were done in triplicate (staurosporine) and duplicate (dasatinib), one sample each of staurosporine and dasatinib were fractionated into 9 fractions using reversedphase chromatography at pH 12 (1 mm Xbridge column, Waters). All samples were dried and acidified prior to LC-MS/ MS analysis. LC-MS/MS Analysis. Samples were dried in vacuo and resuspended in 0.1% formic acid in water. Aliquots of the sample were injected into a nanoACQUITY (Waters) coupled to LTQ-Orbitrap Elite (Thermo-Finnigan). Peptides were separated on custom 50 cm × 75 μM (ID) reversed-phase columns (Reprosil) at 40 °C. Gradient elution was performed from 2% acetonitrile to 40% acetonitrile in 0.1% formic acid over 1−3 h. Orbitrap Elite was operated with XCalibur 2.0/2.1 software. HCD MS/MS spectra were acquired at different nominal resolution settings (15, 30, and 60 K) to determine the optimal setting. For the quantitative experiments, HCD scans were performed at 30 K resolution. For kinobeads experiments, a combination of CID/HCD scans was used32 and the experiments were performed using an inclusion list for kinase peptides as described.33 Peptide and Protein Identification. Mascot 2.0 (Matrix Science) was used for protein identification, using 3 ppm mass tolerance for peptide precursors and 20 mDa (HCD) mass tolerance for fragment ions or 0.6 Da (CID) for CID/HCD experiments. A 2.5 mDa mass tolerance was used for reporter ion extraction in HCD scans by in-house-developed software. Carbamidomethylation of cysteine residues and TMT modification of lysine residues were set as fixed modifications and S,T,Y phosphorylation, methionine oxidation, N-terminal acetylation of proteins and TMT modification of peptide Ntermini were set as variable modifications. The search database consisted of a customized version of the IPI protein sequence database combined with a decoy version of this database created using a script supplied by Matrix Science, the same was done for the E. coli database when searching the E. coli data. Unless stated otherwise, we accepted protein identifications as follows: (i) For single spectrum to sequence assignments, we required this assignment to be the best match and have a minimum Mascot score of 31 and show a 10× difference of this assignment over the next best assignment. Based on these criteria, the decoy search results indicated
