Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples
- ORNL
- National Institutes of Health
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.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- DE-AC05-00OR22725
- OSTI ID:
- 930914
- Journal Information:
- Journal of Proteome Research, Vol. 6, Issue 8
- Country of Publication:
- United States
- Language:
- English
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