TY - JOUR
T1 - Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics
AU - Gerbasi, Vincent R.
AU - Melani, Rafael D.
AU - Abbatiello, Susan E.
AU - Belford, Michael W.
AU - Huguet, Romain
AU - McGee, John P.
AU - Dayhoff, Dawson
AU - Thomas, Paul M.
AU - Kelleher, Neil L.
N1 - Funding Information:
This research was conducted as part of the National Resource for Translational and Developmental Proteomics under Grant No. P41 GM108569 from the National Institute of General Medical Sciences and the National Institutes of Health, under Grant No. UH3 CA246635-02 (N.L.K.).
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/4/27
Y1 - 2021/4/27
N2 - Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
AB - Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
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U2 - 10.1021/acs.analchem.1c00402
DO - 10.1021/acs.analchem.1c00402
M3 - Article
C2 - 33844503
AN - SCOPUS:85105038413
SN - 0003-2700
VL - 93
SP - 6323
EP - 6328
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -