Skip to main content
Publication

Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples

by William J Hervey Iv, Michael B Strader, Michael Strader, Gregory B Hurst
Publication Type
Journal
Journal Name
Journal of Proteome Research
Publication Date
Page Numbers
3054 to 3061
Volume
6
Issue
8

Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high nanogram to low microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the "shotgun" mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 �g portions of protein isolates from two disparate sources: Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters: number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps.