title: Measuring and managing ratio compression for accurate iTRAQ/TMT quantification.
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.
authors.affiliation: Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Mikhail.M.Savitski@gsk.com
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Savitski
authors.forename: Toby
authors.initials: T
authors.lastname: Mathieson
authors.forename: Nico
authors.initials: N
authors.lastname: Zinn
authors.forename: Gavain
authors.initials: G
authors.lastname: Sweetman
authors.forename: Carola
authors.initials: C
authors.lastname: Doce
authors.forename: Isabelle
authors.initials: I
authors.lastname: Becher
authors.forename: Fiona
authors.initials: F
authors.lastname: Pachl
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
authors.forename: Marcus
authors.initials: M
authors.lastname: Bantscheff
mesh_terms: D000465:Algorithms; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods / Q000592:standards*; D013194:Staining and Labeling / Q000379:methods / Q000592:standards*; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments