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.

Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach

“…Mass Spectrometry-LC-MS/MS analyses were performed either on an EasyLC II nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific), or on an EasyLC 1000 nano-UHPLC (Proxeon Biosystems, Odense, Denmark) coupled to a Q Exactive HF mass spectrometer (Thermo Scientific), both as described previously (37,38). Peptide mixtures were injected onto the column in HPLC solvent A (LTQ Orbitrap Elite: 0.5% acetic acid; Q Exactive HF: 0.1% formic acid) at a flow rate of 500 nl/min and subsequently eluted with either a 116 min (PCS analyses) of 5-33-50 -90% of HPLC solvent B (80% acetonitrile in 0.5% acetic acid) or a 57 min gradient (quantification using stable isotope-labeled) of 10 -33-50 -90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid).…”

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

“…For example, two dimensional chromatography fractionation (8,9) and increased reverse phase column length (10,11) were reported to enhance peak capacity for proteome analyses. Improvements in quadrupole performance through higher resolution and scanning rate have enabled more efficient isolation and transmission of target ions with narrow isolation windows in turn reducing chimeric tandem mass spectra (12).…”

A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets

“…The approach scales from small to very large data sets without losing performance, consistently Shotgun proteomics is the most popular approach for large-scale identification and quantification of proteins. The rapid evolution of high-end mass spectrometers in recent years (1)(2)(3)(4)(5) has made proteomic studies feasible that identify and quantify as many as 10,000 proteins in a sample (6 -8) and enables many lines of new scientific research including, for example, the analysis of many human proteomes, and proteome-wide protein-drug interaction studies (9 -11). One fundamental step in most proteomic experiments is the identification of proteins in the biological system under investigation.…”