Implications of partial tryptic digestion in organic–aqueous solvent systems for bottom-up proteome analysis

For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. However, our results demonstrate that tryptic digestion in 80% acetonitrile is less efficient than that of conventional (aqueous) digestion. This is a consequence of decreased enzyme activity beyond ∼40% acetonitrile, increased enzyme autolysis and lower protein solubility in 80% acetonitrile. We observe multiple missed cleavages and reduced concentration of fully cleaved digestion products. Nonetheless we confirm, through room temperature solvent-assisted digestion, a consistent improvement in protein sequence coverage when analyzed by mass spectrometry. These results are explained through the increased number of unique digestion products available for detection. Thus, while solvent-assisted digestion has clear merits for proteome analysis, one should be aware of the inefficiency of protein digestion though this protocol, particularly with absolute protein quantitation experiments.

. While solvent-assisted digestion can rapidly produce high quality protein digestion maps, trypsin digestion efficiency under these conditions is shown to be significantly reduced. High sequence coverage is explain through detection of a large diversity of peptides, though the absolute quantity of these products is far less than a conventional (overnight aqueous) digestion. If a complete (or near complete) protein digestion is desired, solvent-assisted digestion is therefore not recommended.Figure optionsDownload as PowerPoint slideHighlights
► LC/MS, SDS PAGE and BAEE assay are used to assess trypsin activity in 80% acetonitrile
► Above 40% acetonitrile, (unmodified) trypsin activity is lower at 37 °C than at 22 °C
► Low enzyme activity results in poor protein digestion in high organic solvent
► Incomplete digestion can result in higher protein sequence coverage
► High sequence coverage should not be taken as indication of high digestion efficiency