Ion–Ion Charge Reduction Addresses Multiple Challenges Common to Denaturing Intact Mass Analysis

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.