Work

Proton Transfer Charge Reduction Enables Isobaric Labeling-Based Proteoform Quantification of Overlapping Signals in Top-Down Mass Spectrometry

Year: 2026

Type: article

Abstract: Top-down mass spectrometry provides a powerful approach for analyzing and quantifying intact proteoforms, i.e., the distinct molecular forms of proteins. Isobaric labeling-based quantification strategies offer the advantages of multiplexing and increased analytical depth. However, a major challenge remains the quantification of proteoforms when their precursor signals overlap, leading to mixed reporter ion intensities. In this proof-of-concept study, we employed proton transfer charge reduction (PTCR) at the MS2 level to resolve overlapping precursor signals, allowing selective isolation of individual proteoforms and subsequently, their accurate reporter ion quantification at the MS3 level. Using direct infusion mass spectrometry of model proteins labeled with cysteine-directed tandem mass tags, we demonstrate that this approach enables accurate, interference-free reporter ion-based quantification in the presence of overlapping proteoforms and spectrally congested backgrounds. This work highlights PTCR as a versatile gas-phase separation strategy to enhance the quantitative capabilities of labeling-based top-down mass spectrometry, offering a path toward precise, proteoform-resolved quantification across diverse experimental approaches, such as large-scale top-down proteomics.

Source: Journal of the American Society for Mass Spectrometry

DOI: 10.1021/jasms.6c00060

Authors: Philipp T. Kaulich, Andreas Tholey

Institution: Christian-Albrechts-Universität zu Kiel

Language: English

Topic: Advanced Proteomics Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funder: Deutsche Forschungsgemeinschaft