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 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 min 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.
Highlights
Increased proteome coverage with Orbitrap Exploris 480 MS and Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) using single compensation voltages and short LC gradients
We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for dependent acquisition (DDA) and data-independent acquisition (DIA), respectively
The requirement of ultra-high vacuum (UHV) in the 10E-10 or even 10E-11 mbar range for Orbitrap analysis of ions formed at atmospheric pressure has previously necessitated a bulky, multi pump vacuum system, which represented the major constraint on the overall design and size of the instrument
Summary
Increased proteome coverage with Orbitrap Exploris 480 MS and FAIMS using single compensation voltages and short LC gradients. To overcome the precursor selection problem inherent to DDA, data-independent acquisition (DIA) offers systematic measurement of all peptide ions regardless of their intensity by co-fragmenting all co-eluting peptides in broader precursor isolation windows. This provides wider dynamic range of the proteomes analyzed, improved reproducibility for identification and enabled better sensitivity and accuracy for quantification. The highly multiplexed fragment ion spectra in DIA require more elaborate processing algorithms for identification and quantification These typically rely on spectral libraries previously recorded by data-dependent acquisition of similar sample types. From the ‡The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark; §Thermo Fisher Scientific, Bremen, Germany
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