Journal
JOURNAL OF PROTEOME RESEARCH
Volume 13, Issue 12, Pages 6187-6195Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr500985w
Keywords
Orbitrap; Q Exactove HF; shotgun progteomics; HCD; HeLa; phosphoproteomics; single-shot analysis; high-pH reversed-phase fractionation; parallel acquisition; deep progeome coverage
Categories
Funding
- Novo Nordisk Foundation
- Sapere Aude Research Leader grant
- PRIME-XS, a seventh Framework Programme of the European Union [262067-PRIME-XS]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available