Surfactant Cocktail-Aided Extraction/Precipitation/On-Pellet Digestion Strategy Enables Efficient and Reproducible Sample Preparation for Large-Scale Quantitative Proteomics

For
quantitative proteomics, efficient, robust, and reproducible sample
preparation with high throughput is critical yet challenging, especially
when large cohorts are involved, as is often required by clinical/pharmaceutical
studies. We describe a rapid and straightforward surfactant cocktail-aided
extraction/precipitation/on-pellet digestion (SEPOD) strategy to address
this need. Prior to organic solvent precipitation and on-pellet digestion,
SEPOD treats samples with a surfactant cocktail (SC) containing multiple
nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible
protein extraction, including membrane-bound proteins; (ii) effective
removal of detrimental nonprotein matrix components (e.g., >94%
of phospholipids); (iii) rapid/efficient proteolytic digestion owing
to dual (surfactants + precipitation) denaturation. The optimal SC
composition and concentrations were determined by Orthogonal-Array-Design
investigation of their collective/individuals effects on protein extraction/denaturation.
Key parameters for cleanup and digestion were experimentally identified
as well. The optimized SEPOD procedures allowed a rapid 6 h digestion
providing a clean digest with high peptide yields and excellent quantitative
reproducibility (especially low-abundance proteins). Compared with
filter-assisted sample preparation (FASP) and in-solution digestion,
SEPOD showed superior performance by recovering substantially more
peptide/proteins (including integral membrane proteins), yielding
significantly higher peptide intensities and improving quantification
for peptides with extreme physicochemical properties. SEPOD was further
applied in a large-cohort temporal investigation of 44 IAV-infected
mouse lungs, providing efficient and reproducible peptide yields (77.9 ±
4.6%) across all samples. With the IonStar pipeline, >6 400
unique protein groups were quantified (≥2 peptide/protein,
peptide-FDR < 0.05%), ∼99% without missing data in any sample
with <7% technical median-intragroup CV. Altered proteome patterns
revealed interesting novel insights into pathophysiological changes
by IAV infection. In summary, SEPOD offers a feasible solution for
rapid, efficient, and reproducible preparation of biological samples,
facilitating high-quality proteomic quantification of large sample
cohorts.