Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads

Complete, reproducible extraction of protein material
is essential
for comprehensive and unbiased proteome analyses. A current gold standard
is single-pot, solid-phase-enhanced sample preparation (SP3), in which
organic solvent and magnetic beads are used to denature and capture
protein aggregates, with subsequent washes removing contaminants.
However, SP3 is dependent on effective protein immobilization onto
beads, risks losses during wash steps, and exhibits losses and greater
costs at higher protein inputs. Here, we propose solvent precipitation
SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced
protein aggregates by brief centrifugation rather than magnetismwith
optional low-cost inert glass beads to simplify handling. SP4 recovered
equivalent or greater protein yields for 1–5000 μg preparations
and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome
profiling revealed that SP4 yielded a greater recovery of low-solubility
and transmembrane proteins than SP3, benefits to aggregating protein
using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap.
SP4 was verified in three other labs across eight sample types and
five lysis buffersall confirming equivalent or improved proteome
characterization vs SP3. With near-identical recovery,
this work further illustrates protein precipitation as the primary
mechanism of SP3 protein cleanup and identifies that magnetic capture
risks losses, especially at higher protein concentrations and among
more hydrophobic proteins. SP4 offers a minimalistic approach to protein
cleanup that provides cost-effective input scalability, the option
to omit beads entirely, and suggests important considerations for
SP3 applicationsall while retaining the speed and compatibility
of SP3.