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

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. Further exploration of the paramagnetic bead-based approach,
single-pot solid-phase-enhanced sample preparation (SP3), is carried
out toward 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 nondenatured
proteins, and (6) capture of individual proteins from noncomplex mixtures.
These results highlight the robust handling of proteins in a broad
set of scenarios while also enabling the 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.