Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry

Abstract

The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome analysis system, more than 10,000 protein groups were identified from HEK293 cell digests within 120 min of MS measurement time. Additionally, we applied our approach to the analysis of host proteins in mouse feces and detected as many as 892 host protein groups (771 upregulated/121 downregulated proteins) in a mouse model of repeated social defeat stress (R-SDS) used in studying depression. Interestingly, 285 proteins elevated by R-SDS were related to mental disorders. The fecal host protein profiling by deep proteome analysis may help us understand mental illness pathologies noninvasively. Thus, our approach will be helpful for an in-depth comparison of protein expression levels for biological and medical research because it enables the analysis of highly proteome coverage in a single-shot measurement.

Characterization of proteogenomic signatures of differentiation of CD4+ T cell subsets

“…Protein concentration in the protein extract was determined using a BCA protein assay kit (Thermo Fisher Scientific # 23225) and adjusted to 1 μg/μl with 100 mM Tris–HCl (pH 8.5) containing 2% SDS. As previously reported, 18 the 20 µl of protein extract was reduced and alkylated, then digested by trypsin/Lys-C Mix (Promega # V5072), followed by desalting with SDB STAGE tip (GL Sciences Inc. # 7820-11200).…”

“…About 500 ng of peptides was directly injected onto a 75 μm × 20 cm PicoFrit emitter (New Objective # PF360-75-8-N-5) packed in-house with C18 core–shell particles (CAPCELL CORE MP 2.7 μm, 160 Å material; Osaka Soda # 51227 (disassembled the column and got the particles)) at 50°C and then separated with a 120-min gradient at a flow rate of 100 nl/min using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific). Peptides eluting from the column were analysed on a Q Exactive HF-X (Thermo Fisher Scientific) for overlapping window DIA-MS. 18 MS1 spectra were collected in the range of 495-745 m/z at 30,000 resolution to set an automatic gain control (AGC) target of 3e6 and maximum injection time of ‘auto’. MS2 spectra were collected in the range of more than 200 m/z at 45,000 resolution to set an AGC target of 3e6, maximum injection time of ‘auto’, and stepped normalized collision energy of 22%, 26%, and 30%.…”

“…Furthermore, recent advances in liquid chromatography-coupled mass spectrometry in data-independent acquisition mode (DIA-LC–MS/MS) have allowed us to more accurately conduct comparative analysis of mRNA and protein profiles in similar depth. 17 , 18 Thus, we are ready to carry out simultaneous mRNA and protein profiling in depth, which is enough to be called proteogenomic analyses.…”

Proteotranscriptomics – A facilitator in omics research

“…Despite this current limit, increases in proteome coverage can further enhance the comprehensiveness of PTA. Advances in mass-spectrometry instrumentation [78] , [79] , [80] and acquisition methods [81] , [82] , [83] enable increasing measurement depth. The use of specific methodology like removal of high‐abundant proteins or fractionation approaches can split sample complexity across the measurement and are readily implementable [84] .…”

D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply charged precursors

“…Mass spectrometry (MS)-based proteomics has matured in recent years as manifested by the deep MS analyses of the human proteome in which up to 17,100 protein groups (86.5% of the total by estimation) have been identified ( 1, 2 ). However, not every protein is readily identifiable, for example, highly acidic proteins tend to escape MS identification ( 3 ).…”