Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteo...

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Veröffentlicht in:Analytical chemistry (Washington) 2024-02, Vol.96 (7), p.2748-2753
Hauptverfasser: Rogers, Holden T., Melby, Jake A., Ehlers, Lauren E., Fischer, Matthew S., Larson, Eli J., Gao, Zhan, Rossler, Kalina J., Wang, Daojing, Alpert, Andrew J., Ge, Ying
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Sprache:eng
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Zusammenfassung:Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed “small-scale serial Size Exclusion Chromatography” (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.3c05733