Development of a high-pH reversed-phase well plate for peptide fractionation and deep proteome analysis of cells and exosomes
Analytical and Bioanalytical Chemistry(2022)
摘要
The complexity of the proteome often limits the number of identified proteins in the nanoflow LC-MS (nanoLC-MS) analysis of samples. Therefore, peptide fractionation is essential for reducing the sample complexity and improving the proteome coverage. In this study, to achieve high-pH reversed-phase (RP)-well plate fractionation for high-throughput proteomics analysis, C18 particles were coated on a 96-well plate, and the sample-loading processes were optimized for high-pH fractionation. The sample capacity of the high-pH RP-well plate was estimated to be ~6 μg of protein. There were 1.85- and 1.71-fold increases in the number of protein groups and peptides identified, respectively, with high-pH RP-well plate fractionation, compared to those without fractionation. In addition, with alkaline C18 well plate fractionation, exosome markers could be detected using ~1 μg of a protein digest of exosomes by microflow LC-MS (microLC-MS). These results illustrate that high-pH RP-well plate fractionation has superior sensitivity and effectiveness in preparing trace amounts of proteins for deep proteome analysis. Graphical abstract
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关键词
High-pH reversed-phase, Peptide fractionation, Proteomics, 96-well plate, Exosomes
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