MS3-based quantitative proteomics using pulsed-Q dissociation

作者：Cao Zhiyun; Evans Adam R; Robinson Rena A S^*

来源：Rapid Communications in Mass Spectrometry, 2015, 29(11): 1025-1030.

DOI：10.1002/rcm.7192

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 MS3 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-MS3 and PQD-MS3 methods. The most intense fragment ion (termed as HCD-MS3-top ion or PQD-MS3-top ion) or y1 ion (i.e., lysine-TMT tag ion; termed as HCD-MS3-y(1) or PQD-MS3-y(1)) in collision-induced dissociation (CID) MS/MS was selected for MS3.
RESULTS: Calculated protein ratios obtained in HCD-MS3-top ion and PQD-MS3-top ion, HCD-MS3-y(1), and PQD-MS3-y(1) are accurate and PQD-MS3 methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins.
CONCLUSIONS: PQD-MS3 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.

• 出版日期2015-6-15