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Measuring and Managing Ratio Compression for Accurate iTRAQ/TMT Quantification

Open access
Savitski, Mikhail M., Mathieson, Toby, Zinn, Nico, Sweetman, Gavain, Doce, Carola, Becher, Isabelle, Pachl, Fiona, Kuster, Bernhard, Bantscheff, Marcus·2013·Journal of Proteome Research
DOI 10.1021/pr400098r
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Abstract

Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%-60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of E. coli tryptic digests.

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Citations

Cites

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Cited by

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Metadata

Authors
Savitski, M. M., Mathieson, T., Zinn, N., Sweetman, G., Doce, C., Becher, I., Pachl, F., Kuster, B., & Bantscheff, M.
Venue
Journal of Proteome Research
Publisher
American Chemical Society (ACS)
Published
2013-07-02
Volume
12
Issue
8
Pages
3586-3598
ISSN
  • 1535-3893 (ISSN-L)
  • 1535-3907
DOI
10.1021/pr400098r
OpenAlex
W2011710911
Cited by
276
References
12
FWCI
7.14
Type
journal article
Language
en
Open access
green

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