OPTIMIZATION OF PROTEIN PRECIPITATION BEFORE MASS SPECTROMETRY

Abstract

Efficient protein precipitation and resolubilization of the resulting pellet are critical steps in sample preparation for proteomics, directly impacting protein recovery, analytical reproducibility, and downstream mass spectrometry performance. This study systematically investigated how salt identity and precipitation conditions influence protein recovery and reconstitution in acetone-based workflows. A wide range of inorganic salts was evaluated for their effects on precipitation efficiency and compositional selectivity. Proteomic analysis revealed that different salts introduced distinct biases in the precipitated protein fraction—for example, sodium sulfate preferentially precipitated more hydrophobic proteins, while zinc salts enriched more hydrophilic ones. Key precipitation parameters were also assessed for their impact on resolubilization efficiency, including acetone concentration, salt type, drying method, centrifugation time, and mechanical handling. Higher acetone concentrations and shorter processing times enhanced protein redissolution in water. Salts containing zinc and copper significantly hindered resolubilization, likely due to metal–protein coordination, but this effect could be partially reversed by the addition of EDTA. Based on these findings, an optimized protocol was proposed to improve both precipitation yield and resolubilization efficiency. This work provides new insights into the interplay between precipitation chemistry, solvent conditions, and protein properties. The results offer practical guidelines for improving sample preparation protocols in bottom-up proteomics and emphasize the importance of optimizing both precipitation and reconstitution steps to achieve consistent and unbiased protein recovery.