Quantification of proteins using isobaric labeling (tandem mass tag or TMT) starts with the reduction of disulfide bonds in proteins with Dithiothreitol (DTT).
Alkylation with iodoacetamide (IAA) after cystine reduction results in the covalent addition of a carbamidomethyl group that prevents the formation of disulfide bonds. Then, overnight digestion of the proteins using trypsin or trypsin/LyC mixture, Tandem Mass Tag (TMT) labeling on Lysine residues, pooling of all samples and the fractionation of peptide mixture. Finally, LC-MS/MS data acquisition and database search for protein identification and quantification.
Fractionation prior to LC-MS/MS analysis effectively detects low abundance proteins (<100ng/mL), which is the concentration range of most clinical biomarkers.
TMT-based approach allows multiplexing of samples. TMT 6-plex reagents produces a series of different reporter ions with nominal masses from 126 to 131 Da at 1 Da intervals. Several TMT reagents are commercially available including TMTzero, TMT duplex, TMT 6-plex, and TMT 10-plex 1. They have the same chemical structure but contain different numbers and combinations of 13C and 15N isotopes in the reporter group. The overall calibrated intensities of reporter ions equal.
Isobaric labeling reagent (TMT) structure:
a. An amine-specific reactive group – an N-hydroxysuccinimide ester, which reacts with primary amines i.e unblocked N-terminals and lysine side chains.
b. A mass reporter group for quantification – the reporter groups are partially fragmented from the peptide during precursor fragmentation in the mass spectrometer.
c. A mass normalizer group to link the reactive and reporter groups – mass normalizer group ensures that the peptide complexity in the MS1 spectra does not increase with multiplexing.
Unless otherwise noted, every analysis utilizes an MS3-based TMT-centric mass spectrometry method. MS2-based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS3-based TMT can partly rescue 2. To 10 precursors selected for MS2/MS3 analysis.
What are the correction factors used for TMT?
TMT reporter ion signals need to be adjusted to account for isotopic impurities in each TMT variant. For TMT-10plex labeling, different batches of TMT reagents have slightly different isotope impurities that need to be included for database search to correct the reporter ion ratio. The information of isotope impurity can be found in the reagent kit. However, I do not normally correct for isotopic impurities of TMT reagents, as this is typically a «1.2% shift.
Database search for protein identification and quantification:
The resulting TMT-MS3 data (.raw files) are processed using MaxQuant with an integrated Andromeda search engine (v.22.214.171.124). Tandem mass spectra were searched against the Uniprot database.
Download and install MaxQuant
Specification of Various Parameters in MaxQuant (All the other parameters in MaxQuant are set to the default values for processing orbitrap-type data)3:
Raw data pane
Using Load, select all raw files from all batches. Update Experiment (a unique value for each of the samples) and Fraction (if available) column values using set experiment and set fractions buttons.
Group-specific parameters pane
Change Type as Reporter ion MS3 and then select “10plex TMT”. Update isobaric impurities values, if available. For Modifications, select Variable modifications as Oxidation (M), Acetyl (Protein N-term); Deamidation (NQ) and Fixed Modifications as Carbamidomethyl© and, TMT labeled N- terminus and lysine residue. Specify Trypsin/P as a cleavage enzyme using Digestion and allow up to 2 missing cleavages. Set Label-free quantification as None.
Global parameters pane
For Sequences select a reference proteome database Uniprot FASTA file and update Max. peptide mass [Da] as 6000. For Protein quantification select Use only unmodified peptides and a list of modifications such as Oxidation (M), Acetyl (Protein N-term) and Deamidation. Select Match between runs in Identification tab.
Update the following two parameters for MS/MS analyzer:
a. FTMS MS/MS match tolerance: 0.05 Da
b. ITMS MS/MS match tolerance: 0.6 Da
For Folder location select tmp directory (optional).
Further data processing is performed using
Perseus v126.96.36.199 4. The search results in
ProteinGroups.txt generated by MaxQuant are directly processed by Perseus software. MaxQuant reports the TMT-MS3 quantitative relative abundance metrics in the columns titled “Reporter intensity corrected”.
Data normalization and analysis in multiple TMT experimental designs 5
Multiplexed Protein Quantification Using the Isobaric TMT … 6
Isobaric matching between runs and novel PSM-level normalization in MaxQuant … 7
AWS Windows instance for MaxQuant/Perseus
Bąchor, R.; Waliczek, M.; Stefanowicz, P.; Szewczuk, Z. Trends in the Design of New Isobaric Labeling Reagents for Quantitative Proteomics. Molecules 2019, 24, 701. ↩
A. Hogrebe, L. von Stechow, D.B. Bekker-Jensen, B.T. Weinert, C.D. Kelstrup, J.V. Olsen Benchmarking common quantification strategies for large-scale phosphoproteomics Nat. Commun., 9 (2018), p. 1045. ↩