Extending the Compatibility of the SP3 Paramagnetic Bead Processing Approach for Proteomics

Abstract

The diversity in protein and peptide biochemistry necessitates robust protocols and reagents for efficiently handling and enriching these molecules prior to analysis with mass spectrometry (MS) or other techniques. In this study, further exploration of the paramagnetic bead-based approach, Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3), is carried out towards updating and extending previously described conditions and experimental workflows. The SP3 approach was tested in a wide range of experimental scenarios, including: 1. Binding solvents (acetonitrile, ethanol, isopropanol, acetone); 2. Binding pH (acidic vs. neutral); 3. Solvent/lysate ratios (50% - 200%, v/v); 4. Mixing and rinsing conditions (on-rack vs. off-rack rinsing); 5. Enrichment of non-denatured proteins; 6. Capture of individual proteins from non-complex mixtures. These results highlight the robust handling of proteins in a broad set of scenarios, while also enabling development of a modified SP3 workflow that offers extended compatibility. The modified SP3 approach is used in quantitative in-depth proteome analyses to compare it with commercial paramagnetic bead-based HILIC methods (MagReSyn) and across multiple binding conditions (e.g. pH and solvent during binding). Together, these data reveal the extensive quantitative coverage of the proteome possible with SP3 independent of the binding approach utilized. The results further establish the utility of SP3 for the unbiased handling of peptides and proteins for proteomic applications.