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

Romain Huguet, Christopher Mullen, Kristina Srzentić, Joseph B. Greer, Ryan T. Fellers, Vlad Zabrouskov, John E.P. Syka, Neil L. Kelleher, Luca Fornelli*

*Corresponding author for this work

Research output: Contribution to journalArticle

4 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)15732-15739
Number of pages8
JournalAnalytical Chemistry
Issue number24
StatePublished - Dec 17 2019

ASJC Scopus subject areas

  • Analytical Chemistry

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