Project PXD003346

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Ratio accuracy evaluation and correction using tandem mass tags


The multiplexing capabilities of isobaric mass tag based protein quantification, such as Tandem Mass Tags (TMT) or Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) have dramatically increased the scope of Mass Spectrometry (MS) based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample pre-fractionation methods allow for comprehensive study of complex proteomes. However, reporter ion based quantification has often been criticized for limited quantification accuracy due to interference from co-eluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy relying on spiking a 6-protein calibration mixture to the samples subject to relative quantification. Our ratio adjustment approach was evaluated on a large scale TMT 10-plex dataset derived from cancer cell lines with chromosome instability. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity based Label Free Quantification. Comparing the two methods we observed that the isobaric tag based quantification workflow, due to its superior precision, was able to confirm more and smaller protein abundance changes, at a fixed False Positive Rate.

Sample Processing Protocol

B. henselae strains were grown at 35°C for 2 days on Columbia agar supplemented with 5% sheep blood (CBA agar) in a humidified atmosphere containing 5% CO2 as recently specified (Quebatte, M., Dick, M. S., Kaever, V., Schmidt, A. & Dehio, C. Dual input control: activation of the Bartonella henselae VirB/D4 type IV secretion system by the stringent sigma factor RpoH1 and the BatR/BatS two-component system. Mol Microbiol 90, 756–775 (2013)). 109 cells were lysed in 50 μl lysis buffer (8M urea, 0.1% RapiGest, 0.1M ammoniumbicarbonate) and disrupted by two cycles of sonication for 20 seconds (Hielscher Ultrasonicator). Protein concentration was determined by BCA assay (Thermo Fisher Scientific) using a small sample aliquot. Aliquots of cell Llysates/standard protein solutions containing 50 ug of total protein were reduced with 5 mM TCEP for 60 min at 37°C (10 min at 95°C for DOC lysis buffer), alkylated with 10 mM iodoacetamide for 30 min in the dark and quenched with 12.5 mM N-acetyl-cysteine. Proteins were digested by addition of Lys-C (Wako) for 4 h at 37°C (protein to Lys-C ration 100:1), diluted to a final urea concentration of 1.5 6 M (or 1% DOC) with 100 mM ammoniumbicarbonate buffer and further digested by addition of trypsin (Promega) and incubation at 37°C for more that 15 h (protein to Trypsin ratio: 50:1). After digestion, the samples were supplemented with TFA to a final concentration of 0.5% and hydrochloric acidHCl to a final concentration of 50 mM. For samples lysed in DOC containing buffer, the samples were centrifuged at 14 000 rpm for 15 minutes, the peptide containing supernatant removed and employed for further analysis. Peptides were desalted on C18 reversed phase spin columns according to the manufacturer’s instructions (Microspin, Harvard Apparatus), dried under vacuum and stored at -80°C until further processing.

Data Processing Protocol

The acquired raw-files were converted to the mascot generic file (mgf) format using the msconvert tool (part of ProteoWizard, version 3.0.4624 (2013-6-3)). The mgf files were searched, using Mascot (Matrix Science, Version 2.4.0), against a concatenated target-decoy database including forward and reversed sequences of the protein entries included in the of the following databases; Homo sapiens (SwissProt,, release date 30/10/2014, 20625 target sequences), Bartonella henselae (SwissProt,, release date 1/06/2013, 1466 target sequences), a custom made database containing the six Calibration-mix Proteins (uniprot accession numbers: P00489, P02789, P01012, P02666, P00722, B6V3I5, downloaded 22/10/2014) and a set of commonly observed contaminants (381 target sequences provided by the MaxQuant software suit, version The Mascot search criteria were set as follows: 10 ppm precursor ion mass tolerance, 0.02 Da fragment ion mass tolerance, full tryptic specificity required (cleavage after lysine or arginine residues unless followed by proline), maximum 2 missed cleavages, fixed modifications: carbamidomethylation (C) and TMT6plex (K and peptide n-terminus), variable modification: oxidation (M). The database search results were post-processed using the Scaffold Q+ software (version 4.3.2, Proteome Software Inc., Portland, OR). Here, protein scores were calculated using the Protein Prophet algorithm {Nesvizhskii:2003kr}. Next, peptide and protein level identifications were filtered to achieve a maximum False Discovery Rate of 1%, on both levels. The FDR was determined using the Scaffold Local FDR algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. TMT reporter ion intensities were extracted using Scaffold Q+. Protein relative quantification was performed using our in-house developed SafeQuant R package {Glatter:2012ki}. This analysis included multiple steps; adjustment of reporter ion intensities for isotopic impurities according to the manufacturer’s instructions, global data normalization by equalizing the total reporter ion intensity across all channels, summation of reporter ion intensities per protein and channel, calculation of protein abundance ratios and testing for differential abundance using empirical Bayes moderated t-statistics{Smyth:2004cf}. Note that when analyzing the 2-proteome mix dataset global normalization factors were derived from the H.sapiens data subset.


Alexander Schmidt, Proteomics Core Facility
Alexander Schmidt, Biozentrum, Universtiy of Basel, 4056 Basel, Switzerland ( lab head )

Submission Date


Publication Date



    Ahrné E, Glatter T, Viganò C, von Schubert C, Nigg EA, Schmidt A. Evaluation and improvement of quantification accuracy in isobaric mass tag based protein quantification experiments. J Proteome Res. 2016 Jun 27 PubMed: 27345528


Showing 1 - 1 of 1 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 60541 no assay title provided (mzIdentML) 9571 144231 71352 407858 96068