The Small-Protein Enrichment Assay (SPEA) for Analysis of Low Abundance Peptide Hormones in Plasma.

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.