Implications of specific ion effects on salt-induced proteome precipitation in organic solvent

Solvent-based protein precipitation is an integral component of the proteomics workflow, as a means of concentrating and purifying proteins ahead of digestion and mass spectrometry. We previously disclosed the critical importance of ionic species above a minimal concentration to maximize protein precipitation in 80 % acetone; however, the type of salt was not varied. Specific ion effects have been examined for decades, with many exceptions to the classic Hofmeister series reported. Specifically, the relative influence of various salts has been ranked for their capacity to enhance or reverse protein solubility, but the relevance in proteome sample preparation has yet to be characterized.

We hereby demonstrate that specific ion effects have a controlling influence on modulating protein aggregation efficiency in 80 % acetone. Distinct salts facilitate aggregation of proteins from a proteomic mixture to varying degrees in organic solvent. Moreover, we reveal a salt-specific systematic precipitation efficiency bias correlating to intrinsic protein properties. Sodium salts of chloride and sulfate were shown to strongly correlate with the protein's molecular weight and degree of aromaticity, with higher MW and aromaticity enhancing aggregation. Zinc salts of chloride and sulfate, on the other hand favored aggregation of low MW proteins. Proteins with a higher fraction of basic amino acids also showed higher precipitation efficiency for zinc salts, while higher histidine content favored precipitation with ZnCl₂ but not with ZnSO₄.

These results point to a combination of protein-ion, protein-solvent, and ion-solvent interactions influencing their solubility in organic solvent, with differing mechanisms of precipitation being strongly salt dependent, and provide a greater understanding of salt-mediated protein precipitation in organic solvent.