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

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Top-down proteomics (TDP) faces challenges analyzing large proteoforms (>30 kDa) due to instrument limitations and proteome complexity.
• Current TDP methods struggle with high-resolution analysis of intact proteomes in the 30-60 kDa mass range.

Purpose of the Study:

• To introduce a novel data acquisition method, proton transfer charge reduction (PTCR), for high-throughput investigation of large proteoforms.
• To enhance the characterization of proteoforms >30 kDa in *Pseudomonas aeruginosa*.

Main Methods:

• A targeted data acquisition strategy (tPTCR) employing two gas-phase fractionation steps was designed.
• The method utilizes a quadrupole filter for charge state selection followed by PTCR to reduce average charge states for simplified mass analysis.
• Intact mass and collisional dissociation data were combined for proteoform identification via database searching.

Main Results:

• The tPTCR method generated proteoform mass spectra with minimal m/z overlap and clear charge state distributions, simplifying mass calculations.
• Benchmarking against existing methods demonstrated that tPTCR doubled the number of identified UniProt entries and proteoforms >30 kDa.
• The method achieved this improvement within the standard liquid chromatography time scale.

Conclusions:

• The proposed PTCR method significantly advances the capability of top-down proteomics for analyzing large and complex proteoforms.
• tPTCR offers a high-throughput and efficient approach for deep proteome characterization, particularly for bacterial proteoforms.
• This technique overcomes previous limitations in high-resolution FTMS analysis of larger protein species.