Abstract
Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.
Highlights
Optimal proteome characterization by mass spectrometry is critically dependent on the successful isolation and recovery of high-purity samples ahead of analysis
We concluded that the combination of higher levels of acetone (97%), together with specific salt types (e.g., ZnSO4), would facilitate the recovery of low mass peptides resulting from tryptic digestion [3]
Intact protein recovery exceeds 90% when precipitated with NaCl, though these larger components remain fully soluble in 80% acetone when the solvent is added below a critical solution ionic strength [3]
Summary
Optimal proteome characterization by mass spectrometry is critically dependent on the successful isolation and recovery of high-purity samples ahead of analysis. We concluded that the combination of higher levels of acetone (97%), together with specific salt types (e.g., ZnSO4), would facilitate the recovery of low mass peptides resulting from tryptic digestion [3] In these experiments, test samples were subject to desalting prior to precipitation to eliminate the potential of the solution matrix interfering with the isolation of peptides while disclosing the role of specific salt types and concentrations. Varying the salt and solvent concentrations gives rise to sigmoidal recovery trends, with an optimal yield obtained at above 100 mm salt and 90% solvent This protocol suggests a convenient approach to quench and recover peptide-digested proteins ahead of MS analysis, providing maximal higher protein sequence coverage when subject to bottom-up MS analysis
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