Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster.

TitleAccurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster.
Publication TypeJournal Article
Year of Publication2012
AuthorsWühr, M, Haas, W, McAlister, GC, Peshkin, L, Rad, R, Kirschner, MW, Gygi, SP
JournalAnal Chem
Volume84
Issue21
Pagination9214-21
Date Published2012 Nov 06
ISSN1520-6882
KeywordsHumans, Mass Spectrometry, Peptide Fragments, Proteomics
Abstract

<p>Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion-ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low m/z reporter ions, the localization of these complement TMT (TMT(C)) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT(C) ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT(C) ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame.</p>

DOI10.1021/ac301962s
Alternate JournalAnal. Chem.
PubMed ID23098179
PubMed Central IDPMC3771681
Grant ListGM026875 / GM / NIGMS NIH HHS / United States
R01 GM026875 / GM / NIGMS NIH HHS / United States
HG3456 / HG / NHGRI NIH HHS / United States
R01 GM067945 / GM / NIGMS NIH HHS / United States
R01 HG003456 / HG / NHGRI NIH HHS / United States
GM67945 / GM / NIGMS NIH HHS / United States