期刊
JOURNAL OF PROTEOME RESEARCH
卷 13, 期 12, 页码 6187-6195出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr500985w
关键词
Orbitrap; Q Exactove HF; shotgun progteomics; HCD; HeLa; phosphoproteomics; single-shot analysis; high-pH reversed-phase fractionation; parallel acquisition; deep progeome coverage
资金
- Novo Nordisk Foundation
- Sapere Aude Research Leader grant
- PRIME-XS, a seventh Framework Programme of the European Union [262067-PRIME-XS]
Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 mu g of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140?000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.
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