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.
ASJC Scopus subject areas
- Analytical Chemistry