Analytica Chimica Acta

Mass spectrometry profiling of low molecular weight proteins and peptides isolated by acetone precipitation

by Venus Baghalabadi & Alan A. Doucette

Solvent-based protein precipitation provides exceptional recovery, particularly when the ionic strength of the solution is controlled. While precipitation is ideally suited for intact protein purification ahead of mass-spectrometry, low molecular weight (LMW) proteins and peptides are considered less susceptible to aggregation in organic solvent. As the combination of salt and organic solvent (i.e. acetone) has yet to be exploited to precipitate LMW proteins, we herein determine the low mass limit for solvent-based protein precipitation. We establish optimized conditions for high recovery precipitation of LMW proteins and peptides. Our results demonstrate a strong dependence on the type of salt to recover LMW components from complex mixtures. Inclusion of 100 mM ZnSO₄ with 97% acetone provides near quantitative recovery of all peptides down to 2 kDa, and continues to exceed 90% yield for peptides at a molecular weight of 1 kDa. A detailed characterization of the precipitated peptides resulting from trypsin and pepsin digestion of complex systems is provided by bottom-up mass spectrometry.