Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Clean-up Techniques.

ABSTRACT: The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identifications. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. Recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. We hypothesize that differences in capture mechanisms of SP3 and SP4 affect which proteins are identified by each clean-up technique. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. Ultimately, we demonstrate that protein-capture mechanisms are distinct, and selection of a clean-up technique that yields high proteome coverage is dependent on protein-sample hydrophobicity. Data has been deposited into MassIVE (MSV000094130) and ProteomeXchange (PXD049965).

INSTRUMENT(S): Synapt G2-S HDMS, nanoACQUITY UPLC

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Elyssia Gallagher  

PROVIDER: MSV000094130 | MassIVE | Tue Feb 20 08:11:00 GMT 2024

SECONDARY ACCESSION(S): PXD049965

REPOSITORIES: MassIVE