Contents

Interference-Staged Charge-State Evidence Partitioning for MALDI MS/MS Identification of Peptides, Intact Proteins, and Lysate Components

Author(s): Louis E. Brus1
1Department of Chemistry, Columbia University, New York, New York 10027, USA
Louis E. Brus
Department of Chemistry, Columbia University, New York, New York 10027, USA

Abstract

Co-isolation in MALDI tandem mass spectrometry is conventionally considered spectral contamination; yet, the acquisition information can contain evidence of charge-state continuity, shared precursor windows, and analyte-specific fragment retention. In this paper, we introduce Charge-State Evidence Partitioning (CSEP) as an interference-aware analytical workflow to discriminate between an ambiguous overlap that requires spectrum rejection as an unresolved case and a meaningful multi-analyte measurement with recoverable fragments. The present investigation uses seven channels for analytes that span three levels of complexity: a pair of ACTH 1-17 and angiotensin II peptides, a pair of ubiquitin and cytochrome C proteins, and a tripartite mixture of bradykinin, myoglobin, and \textit{Escherichia coli} lysate. The research question is formulated narrowly: can evidence from a shared precursor window be routed into the relevant charge-state family and used to search the database only if its location suggests such an operation to be justified? CSEP applies a window registry, charge-state routing, fragment recurrence evaluation, and cross-channel evidence specificity test before database search results are generated. The score outcome depended on the analyte: ACTH 1-17 from 71 up to 153, angiotensin II from 25 up to 66, ubiquitin from 42 up to 288, bradykinin from 3 up to 56, and DNA-binding protein HU-alpha from 55 up to 254; the cytochrome C and myoglobin were considered separate protein channels with respective scores of 225 and 149. Analyte score improvement was significant especially in cases when the protection against contamination provided by several charge-state windows enabled detection of a weak fragment signal in a crowded mixed window.

Keywords: MALDI MS/MS; chimeric spectra; charge-state continuity; precursor co-isolation; top-down proteomics; peptide identification; protein identification; analytical spectroscopy.
Copyright © 2025 Louis E. Brus. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.