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

Abstract

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.

The Cannabis Proteome Draft Map Project

“…Multiple variations on protein extraction and digestion were tested, based on the highest percent recovery of peptides per milligram of starting plant material by use of an absorbance (562 nm) assay for tryptic peptides (Pierce BCA Protein Assay Kit Cat# 23225) (data not shown). The final sample method was based on the filter-aided sample preparation method (FASP, Expedeon SKU:44250) [37]. Briefly, 1 mg of fresh plant flower was flash frozen at −80 • C for 20 min.…”

MS1 ion current‐based quantitative proteomics: A promising solution for reliable analysis of large biological cohorts

“…SEPOD utilizes a high‐concentration surfactant cocktail (SC) buffer containing multiple non‐ionic/anionic surfactants (e.g., SDS, SDC, IGEPAL CA‐630), which are then removed by precipitation with organic solvent (e.g., acetone). The detergent cocktail achieves three important goals: (i) exhaustive/reproducible protein extraction, including MPs from cells and tissues; for example, the SC buffer significantly outcompeted SDT buffer in protein extraction from lung and brain, suggesting the use of multiple surfactants may afford more efficient disruption of cellular compartments and thereby enhancing protein extraction from tissues (Shen et al, ); (ii) effective removal of detrimental non‐protein matrix components (e.g., fatty acids, phospholipids, etc.) which would otherwise compromise the robustness of digestion and LC‐MS analysis; (iii) highly effective proteolytic digestion owing to the through, dual‐action (surfactants + precipitation) denaturation.…”

“…which would otherwise compromise the robustness of digestion and LC‐MS analysis; (iii) highly effective proteolytic digestion owing to the through, dual‐action (surfactants + precipitation) denaturation. Compared with FASP and in‐solution digestion, SEPOD showed substantially higher peptide/protein (including MPs) recovery and ∼20–40% more peptide identifications, as well as improved quantification of peptides with extreme physicochemical properties in large sample cohorts (Shen et al, ). More importantly, the procedure enabled highly efficient, reproducible and robust preparation of large sample cohorts, as exemplified by analysis of 44 lung tissue samples in a time‐course investigation post virus infection (Shen et al, ).…”

The Cannabis Multi-Omics Draft Map Project

“…The final sample method was based on the filter-aided sample preparation method (FASP). 14 Briefly, 1mg of fresh plant material was flash frozen at -80°C for 20 minutes. The cell walls were disrupted by immediately removing the frozen material and blunt physical concussion.…”