A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

Abstract

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 unique peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 minute LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins. The authors would like to thank Stoyan Stoychev and Justin Jordaan for great help and input for optimizing our phospho-enrichment workflow and establishing automated platform for sample preparation. We would also like to thank Nicolai Bache for help and input on the chromatographic methods as well as Anna Secher for providing rat tissues and all members of the Olsen Group for critical input on the manuscript.

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

“…The innovations on MS instrumentation, labeling methodologies and sample preparation strategies are expected to overcome some of the abovementioned limitations, and further improve detection sensitivity in single-cell proteome analysis. For example, the combination of iBASIL with nanoscale fractionation 10 can further improve the proteome coverage while reducing the precursor co-isolation; the newly available Real Time Search-MS3 method (RTS-MS3) 28 or ion mobility technique [30][31] maybe a solution for precise and accurate quantitation without sacrificing the proteome coverage.…”

Deep learning the collisional cross sections of the peptide universe from a million training samples

“…The combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) extends conventional liquid chromatography-mass spectrometry by an extra dimension of separation, increasing peak capacity, selectivity and depth of analysis [1][2][3][4][5] . Recent advances have greatly improved the sensitivity of commercially available IMS devices and the technology is now set for a broader application in MSbased proteomics [6][7][8][9][10] .…”

Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

“…The normal SWATH method is based on a short gradient SWATH method published recently 10 , with slightly modified cycle time and precursor range to match the parameters of the scanning SWATH method, to allow direct comparison (see Methods). The data was further compared to recently published 5-minute gradient DIA runs of cell lysates (HeLa) acquired on Orbitrap Exploris 480 with a FAIMS coupled to an Evosep One system ("5min DIA FAIMS") (PXD016662) 19 . In order to make the identification numbers comparable, the runs were analyzed with project-specific libraries generated on the respective setups using DIA-NN (see Methods).…”

“…Further, fast, efficient and robust chromatographic separations have been achieved by replacing nanoflow LC, as traditionally used in proteomics 13,14 , with setups that use higher flow-rates. This ranges from microflow LC systems (5-50 µ l/min) [15][16][17] , to novel LC devices with preformed gradients 18,19 . More recently, we have introduced proteome experiments that make use of high-flow liquid chromatography (800 µl/min).…”