Coupling high-field asymmetric ion mobility spectrometry with capillary electrophoresis-electrospray ionization-tandem mass spectrometry improves protein identifications in bottom-up proteomic analysis of low nanogram samples
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ABSTRACT: In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultra-sensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages (CVs) per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng HeLa protein digest standard identified 1,314±30 proteins, 4,829±200 peptide groups, and 7,577±163 peptide spectrum matches (PSMs) corresponding to a 16%, 25%, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Our results also identified from 1 ng HeLa protein digest without any prior enrichment, 76±9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hela Cell
SUBMITTER: Kendall Johnson
LAB HEAD: Alexander R. Ivanov
PROVIDER: PXD034255 | Pride | 2023-05-10
REPOSITORIES: Pride
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