TY - JOUR
T1 - Ion-Ion Charge Reduction Addresses Multiple Challenges Common to Denaturing Intact Mass Analysis
AU - Bailey, Aaron O.
AU - Huguet, Romain
AU - Mullen, Christopher
AU - Syka, John E.P.
AU - Russell, William K.
N1 - Funding Information:
We thank Prof. Matthieu Gagnon (University of Texas Medical Branch) for the kind gift of the purified IF2 sample. The UTMB Mass Spectrometry Facility receives support from the Cancer Prevention Research Institute of Texas (CPRIT) grant no RP190682.
Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022/3/8
Y1 - 2022/3/8
N2 - Complete LC-MS-based protein primary sequence characterization requires measurement of intact protein profiles under denaturing and/or reducing conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we propose the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR"(fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. Denaturing analysis of the flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced overcharging and showed an 11-fold gain in S/N compared to conventional MS1. Analysis by fsPTCR-MS of the microheterogeneous glycoprotein fetuin revealed twice as many proteoforms as MS1 (112 vs 56). In a macroheterogeneous mixture of proteins ranging from 14 to 148 kDa, fsPTCR provided more than 10-fold increased sensitivity and quantitative accuracy for diluted bovine serum albumin (66 kDa). Finally, our analysis shows that collisional gas pressure is a key parameter which can be utilized during fsPTCR to retain or remove larger proteins from acquired spectra.
AB - Complete LC-MS-based protein primary sequence characterization requires measurement of intact protein profiles under denaturing and/or reducing conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we propose the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR"(fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. Denaturing analysis of the flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced overcharging and showed an 11-fold gain in S/N compared to conventional MS1. Analysis by fsPTCR-MS of the microheterogeneous glycoprotein fetuin revealed twice as many proteoforms as MS1 (112 vs 56). In a macroheterogeneous mixture of proteins ranging from 14 to 148 kDa, fsPTCR provided more than 10-fold increased sensitivity and quantitative accuracy for diluted bovine serum albumin (66 kDa). Finally, our analysis shows that collisional gas pressure is a key parameter which can be utilized during fsPTCR to retain or remove larger proteins from acquired spectra.
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U2 - 10.1021/acs.analchem.1c04973
DO - 10.1021/acs.analchem.1c04973
M3 - Article
AN - SCOPUS:85125636500
SN - 0003-2700
VL - 94
SP - 3930
EP - 3938
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 9
ER -