Performance Evaluation of the Q Exactive HF‑X for Shotgun Proteomics
journal contributionposted on 29.11.2017, 00:00 by Christian D. Kelstrup, Dorte B. Bekker-Jensen, Tabiwang N. Arrey, Alexander Hogrebe, Alexander Harder, Jesper V. Olsen
Progress in proteomics is mainly driven by advances in mass spectrometric (MS) technologies. Here we benchmarked the performance of the latest MS instrument in the benchtop Orbitrap series, the Q Exactive HF-X, against its predecessor for proteomics applications. A new peak-picking algorithm, a brighter ion source, and optimized ion transfers enable productive MS/MS acquisition above 40 Hz at 7500 resolution. The hardware and software improvements collectively resulted in improved peptide and protein identifications across all comparable conditions, with an increase of up to 50 percent at short LC–MS gradients, yielding identification rates of more than 1000 unique peptides per minute. Alternatively, the Q Exactive HF-X is capable of achieving the same proteome coverage as its predecessor in approximately half the gradient time or at 10-fold lower sample loads. The Q Exactive HF-X also enables rapid phosphoproteomics with routine analysis of more than 5000 phosphopeptides with short single-shot 15 min LC–MS/MS measurements, or 16 700 phosphopeptides quantified across ten conditions in six gradient hours using TMT10-plex and offline peptide fractionation. Finally, exciting perspectives for data-independent acquisition are highlighted with reproducible identification of 55 000 unique peptides covering 5900 proteins in half an hour of MS analysis.
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40 Hzbenchtop Orbitrap series7500 resolution50 percentpredecessorhalfTMT 10-plexoptimized ion transfersprotein identificationsproteomics applicationsPerformance Evaluationgradient hours16 70016 700 phosphopeptidesMS instrumentShotgun Proteomics ProgressMS analysisproteome coveragedata-independent acquisitionsoftware improvementsQ Exactive HF-XLCsample loadsgradient timepeak-picking algorithmoffline peptide fractionation5900 proteins55 000ion source5000 phosphopeptidesidentification rates