Language
English
Publication Date
10-1-2025
Journal
Proteomics
DOI
10.1002/pmic.70037
PMID
40904101
PMCID
PMC12491930
PubMedCentral® Posted Date
September 2025
PubMedCentral® Full Text Version
Post-print
Abstract
Mass spectrometry (MS)-based single-cell proteomics, while highly challenging, offers unique potential for a wide range of applications to interrogate cellular heterogeneity, trajectories, and phenotypes at a functional level. We report here the development of the spectral library-based multiplex segmented selected ion monitoring (SLB-msSIM) method, a conceptually unique approach with significantly enhanced sensitivity and robustness for single-cell analysis. The single-cell MS data is acquired by a multiplex segmented selected ion monitoring (msSIM) technique, which sequentially applies multiple isolation cycles with the quadrupole using a wide isolation window in each cycle to accumulate and store precursor ions in the C-trap for a single scan in the Orbitrap. Proteomic identification is achieved through spectral matching using a well-defined spectral library. We applied the SLB-msSIM method to interrogate cellular heterogeneity in various pancreatic cancer cell lines, revealing common and distinct functional traits among PANC-1, MIA-PaCa2, AsPc-1, HPAF, and normal HPDE cells. Furthermore, for the first time, our novel data revealed the diverse cell trajectories of individual PANC-1 cells during the induction and reversal of epithelial-mesenchymal transition (EMT). Collectively, our results demonstrate that SLB-msSIM is a highly sensitive and robust platform, applicable to a wide range of instruments for single-cell proteomic studies. SUMMARY: We present the SLB-msSIM method, a conceptually unique approach in mass spectrometry-based single-cell proteomics that significantly enhances sensitivity and robustness. This innovative platform enables detailed analysis of the proteome landscape, capturing cellular heterogeneity, trajectories, and phenotypes at a single-cell resolution. Utilizing the SLB-msSIM technique, we identified both common and distinct functional traits among various pancreatic cancer cell lines and normal cells. Moreover, our study unveiled new insights into the diverse cell trajectories of individual cancer cells during the induction and reversal of epithelial-mesenchymal transition (EMT). In summary, the SLB-msSIM method offers a highly sensitive and robust platform for single-cell proteomic studies, with broad applicability across different instruments.
Keywords
Single-Cell Analysis, Humans, Proteomics, Cell Line, Tumor, Pancreatic Neoplasms, Mass Spectrometry, Proteome, cancer cell heterogeneity, epithelial‐mesenchymal transition (EMT), mass spectrometry, proteomics, single‐cell proteomics, spectral library‐based multiplex segmented selected ion monitoring (SLB‐msSIM)
Published Open-Access
no
Recommended Citation
Senavirathna, Lakmini; Ma, Cheng; Duong, Van-An; et al., "SLB-msSIM: A Spectral Library-Based Multiplex Segmented SIM Platform for Single-Cell Proteomic Analysis" (2025). The Brown Foundation: Institute of Molecular Medicine. 36.
https://digitalcommons.library.tmc.edu/molecular_med/36