Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms

Abstract

Despite the recent technological advances in Fourier transform mass spectrometry (FTMS) instrumentation, top-down proteomics (TDP) is currently mostly applied to the characterization of proteoforms <30 kDa due to the poor performance of high-resolution FTMS for the analysis of larger proteoforms and the high complexity of intact proteomes in the 30-60 kDa mass range. Here, we propose a novel data acquisition method based on ion-ion proton transfer, herein termed proton transfer charge reduction (PTCR), to investigate large proteoforms of <i>Pseudomonas aeruginosa</i> in a high-throughput fashion. We designed a targeted data acquisition strategy, named tPTCR, which applies two consecutive gas phase fractionation steps for obtaining intact precursor masses: first, a narrow (1.5 <i>m</i>/<i>z</i>-wide) quadrupole filter <i>m</i>/<i>z</i> transmission window is used to select a subset of charge states from all ionized proteoform cations; second, this aliquot of protein cations is subjected to PTCR in order to reduce their average charge state: upon <i>m</i>/<i>z</i> analysis in an Orbitrap, proteoform mass spectra with minimal <i>m</i>/<i>z</i> peak overlap and easy-to-interpret charge state distributions are obtained, simplifying the proteoform mass calculation. Subsequently, the same quadrupole-selected narrow <i>m</i>/<i>z</i> region of analytes is subjected to collisional dissociation to obtain proteoform sequence information, which used in combination with intact mass information leads to proteoform identification through an off-line database search. The newly proposed method was benchmarked against the previously developed "medium/high" data-dependent acquisition strategy and doubled the number of UniProt entries and proteoforms >30 kDa identified on the liquid chromatography time scale.