FLASHQuant: A Fast Algorithm for Proteoform Quantification in Top-Down Proteomics

Accurate quantification of individual proteoforms is a crucial step in identifying proteome-wide alterations in different biological conditions. Intact proteoforms have been analyzed predominantly by liquid chromatography–mass spectrometry (LC-MS)-based top-down proteomics (TDP) and quantified prima...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2024-10, Vol.96 (43), p.17227-17234
Main Authors: Kim, Jihyung, Jeong, Kyowon, Kaulich, Philipp T., Winkels, Konrad, Tholey, Andreas, Kohlbacher, Oliver
Format: Article
Language:English
Subjects:
Algorithms
Chromatography, Liquid - methods
Humans
Labels
Liquid chromatography
Mass spectrometry
Mass Spectrometry - methods
Mass spectroscopy
Open source software
Proteome - analysis
Proteomes
Proteomics
Proteomics - methods
Software
Tandem Mass Spectrometry - methods
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Summary:Accurate quantification of individual proteoforms is a crucial step in identifying proteome-wide alterations in different biological conditions. Intact proteoforms have been analyzed predominantly by liquid chromatography–mass spectrometry (LC-MS)-based top-down proteomics (TDP) and quantified primarily by the label-free quantification (LFQ) method, as it requires no additional costly labeling. In TDP, due to frequent coelution and complex signal structures, overlapping signals deriving from multiple proteoforms complicate accurate quantification. Here, we introduce FLASHQuant for MS1-level LFQ analysis in TDP, which is capable of automatically resolving and quantifying coeluting proteoforms. In benchmark tests performed with both spike-in proteins and proteome-level mixture data sets, FLASHQuant was shown to perform highly accurate and reproducible quantification in short runtimes of just a few minutes per LC-MS run. In particular, it was demonstrated that resolving overlapping proteoforms boosts the quantification accuracy. FLASHQuant is publicly available as platform-independent open-source software at https://openms.org/flashquant/, accompanied by the simple alignment algorithm ConsensusFeatureGroupDetector for multiple LC-MS runs.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.4c03117