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bioRxiv · 10.1101/2024.02.14.579877

Gel-Assisted Proteome Position Integral Shift (GAPPIS) Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Novel Caspase 3 Substrates

Meng, Z.·Saei, A. A.·Zhang, X.·Lyu, H.·Gharibi, H.·Lundstrom, S. L.·Vegvari, A.·Gaetani, M.·Zubarev, R. A.
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Abstract

Here we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics, and demonstrate its utility in studying proteolytic events in programmed cell death. With Gel-Assisted Proteome Position Integral Shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: 1) a negative MW shift; 2) an elevated ratio in a pair of a semi-tryptic and tryptic peptide, 3) a negative shift in the standard deviation of MW estimated for different peptides, and 4) a negative shift in skewness of the same data. Of these proteins, 58 molecules were novel caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

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Meng, Z., Saei, A. A., Zhang, X., Lyu, H., Gharibi, H., Lundstrom, S. L., Vegvari, A., Gaetani, M., Zubarev, R. A.. 2024-02-15. Gel-Assisted Proteome Position Integral Shift (GAPPIS) Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Novel Caspase 3 Substrates. https://doi.org/10.1101/2024.02.14.579877

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