Differences\nin Protein Capture by SP3 and SP4 Demonstrate\nMechanistic Insights of Proteomics Cleanup Techniques

The goal of proteomics experiments is to identify proteins\nto observe\nchanges in cellular processes and diseases. One challenge in proteomics\nis the removal of contaminants following protein extraction, which\ncan limit protein identifications. Single-pot, solid-phase-enhanced\nsample preparation (SP3) is a cleanup technique in which proteins\nare captured on carboxylate-modified particles through a proposed\nhydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism.\nRecent results have suggested that proteins are captured in SP3 due\nto a protein-aggregation mechanism. Solvent precipitation, single-pot,\nsolid-phase-enhanced sample preparation (SP4) is a newer cleanup technique\nthat employs protein aggregation to capture proteins without modified\nparticles. We hypothesize that differences in capture mechanisms of\nSP3 and SP4 affect which proteins are identified by each cleanup technique.\nHerein, we assess the proteins identified and enriched using SP3 versus\nSP4 for MCF7 subcellular fractions and correlate protein capture in\neach method to protein hydrophobicity. Our results indicate that SP3\ncaptures more hydrophilic proteins through a combination of HILIC-like\nand protein-aggregation mechanisms, while SP4 captures more hydrophobic\nproteins through a protein-aggregation mechanism. Ultimately, we demonstrate\nthat protein-capture mechanisms are distinct, and the selection of\na cleanup technique that yields high proteome coverage is dependent\non protein-sample hydrophobicity. Data has been deposited into MassIVE\n(MSV000094130) and ProteomeXchange (PXD049965).