title: Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.
abstract: 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 μg 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.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Christian D
authors.initials: CD
authors.lastname: Kelstrup
authors.forename: Rosa R
authors.initials: RR
authors.lastname: Jersie-Christensen
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.forename: Tabiwang N
authors.initials: TN
authors.lastname: Arrey
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuehn
authors.forename: Markus
authors.initials: M
authors.lastname: Kellmann
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
mesh_terms: D019985:Benchmarking / Q000379:methods; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein / Q000379:methods
chemical_list: D010455:Peptides; D020543:Proteome
keywords: HCD; HeLa; Orbitrap; Q Exactive HF; deep proteome coverage; high-pH reversed-phase fractionation; parallel acquisition; phosphoproteomics; shotgun proteomics; single-shot analysis