Premium
MS 3 ‐based quantitative proteomics using pulsed‐Q dissociation
Author(s) -
Cao Zhiyun,
Evans Adam R.,
Robinson Renã A. S.
Publication year - 2015
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7192
Subject(s) - chemistry , tandem mass spectrometry , ion , isobaric labeling , isobaric process , mass spectrometry , tandem mass tag , dissociation (chemistry) , fragmentation (computing) , analytical chemistry (journal) , orbitrap , collision induced dissociation , chromatography , quantitative proteomics , proteomics , protein mass spectrometry , biochemistry , physics , organic chemistry , gene , computer science , thermodynamics , operating system
Rationale Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high‐throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co‐isolation and co‐fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS 3 method using pulsed‐Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins. Methods Mouse brain protein digests were labeled with TMT‐128, 129, 130, 131 reagents, mixed in the following molar ratios 1:1:2:5, respectively, and analyzed using HCD‐MS 3 and PQD‐MS 3 methods. The most intense fragment ion (termed as HCD‐MS 3 ‐top ion or PQD‐MS 3 ‐top ion) or y 1 ion (i.e., lysine‐TMT tag ion; termed as HCD‐MS 3 ‐ y 1 or PQD‐MS 3 ‐ y 1 ) in collision‐induced dissociation (CID) MS/MS was selected for MS 3 . Results Calculated protein ratios obtained in HCD‐MS 3 ‐top ion and PQD‐MS 3 ‐top ion, HCD‐MS 3 ‐ y 1 , and PQD‐MS 3 ‐ y 1 are accurate and PQD‐MS 3 methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins. Conclusions PQD‐MS 3 methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.