The analysis of low abundance peptide hormones such as insulin in blood plasma is difficult with unbiased mass spectrometry-based proteomics, as they are overshadowed by very abundant proteins such as albumin and IgG. The small-protein enrichment assay (SPEA) can greatly increase detection and discovery of these factors through specific enrichment, which enables fast and efficient analysis of many small-protein factors using a single untargeted LC-MS/MS acquisition. SPEA uses an alcohol-acid-based dissociation and precipitation step, prior to denaturing SEC to remove the large highly abundant plasma proteins leaving only a small-protein fraction. This is followed by an efficient sample preparation and cleanup before either data-dependent acquisition (DDA), or data-independent acquisition (DIA), LC-MS/MS analysis. Combining these workflows increases discovery of proteins, posttranslational modifications (PTMs), and cleavage sites using DDA, while DIA provides consistent analysis useful for large cohort analysis.
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