Proton Transfer Charge Reduction Enables Isobaric Labeling-Based Proteoform Quantification of Overlapping Signals in...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down mass spectrometry is crucial for intact proteoform analysis and quantification.
• Isobaric labeling enhances multiplexing and analytical depth in proteomics.
• Overlapping precursor signals hinder accurate proteoform quantification.

Purpose of the Study:

• To introduce and validate proton transfer charge reduction (PTCR) at the MS2 level for resolving overlapping proteoform signals.
• To enable selective isolation and accurate quantification of individual proteoforms.
• To improve the quantitative capabilities of labeling-based top-down mass spectrometry.

Main Methods:

• Utilized proton transfer charge reduction (PTCR) at the MS2 level.
• Employed cysteine-directed tandem mass tags for labeling model proteins.
• Performed direct infusion mass spectrometry analysis.
• Achieved MS3-level reporter ion quantification after selective isolation.

Main Results:

• PTCR successfully resolved overlapping precursor signals from proteoforms.
• Accurate, interference-free reporter ion quantification was achieved.
• Demonstrated effectiveness in spectrally congested backgrounds and with overlapping proteoforms.
• Validated the approach using model proteins and direct infusion MS.

Conclusions:

• PTCR is a versatile gas-phase separation strategy for top-down mass spectrometry.
• Enhances quantitative capabilities, enabling proteoform-resolved analysis.
• Offers a pathway for precise quantification in diverse proteomic applications.