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

Anal Chem. 2019 Dec 17;91(24):15732-15739. doi: 10.1021/acs.analchem.9b03925. Epub 2019 Nov 22.

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 Pseudomonas aeruginosa 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 m/z-wide) quadrupole filter m/z 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 m/z analysis in an Orbitrap, proteoform mass spectra with minimal m/z peak overlap and easy-to-interpret charge state distributions are obtained, simplifying the proteoform mass calculation. Subsequently, the same quadrupole-selected narrow m/z 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / metabolism*
  • Chromatography, Liquid / methods*
  • Protein Isoforms
  • Proteome / analysis*
  • Protons*
  • Pseudomonas aeruginosa / metabolism*
  • Software*
  • Tandem Mass Spectrometry / methods*

Substances

  • Bacterial Proteins
  • Protein Isoforms
  • Proteome
  • Protons