Efficient and Specific Trypsin Digestion of Microgram to Nanogram Quantities of Proteins in Organic-Aqueous Solvent Systems
- ORNL
Mass spectrometry-based identification of the components of multiprotein complexes often involves solution-phase proteolytic digestion of the complex. The affinity purification of individual protein complexes often yields nanogram to low-microgram amounts of protein, which poses several challenges for enzymatic digestion and protein identification. We tested different solvent systems to optimize trypsin digestions of samples containing limited amounts of protein for subsequent analysis by LC-MS-MS. Data collected from digestion of 10-, 2-, 1-, and 0.2- g portions of a protein standard mixture indicated that an organicaqueous solvent system containing 80% acetonitrile consistently provided the most complete digestion, producing more peptide identifications than the other solvent systems tested. For example, a 1-h digestion in 80% acetonitrile yielded over 52% more peptides than the overnight digestion of 1 g of a protein mixture in purely aqueous buffer. This trend was also observed for peptides from digested ribosomal proteins isolated from Rhodopseudomonas palustris. In addition to improved digestion efficiency, the shorter digestion times possible with the organic solvent also improved trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent. The technique was also demonstrated for an affinityisolated protein complex, GroEL. To our knowledge, this report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity. Mass spectrometry (MS) has become a widely used method for studying proteins, protein complexes, and whole proteomes because of innovations in soft ionization techniques, bioinformatics, and chromatographic separation techniques.1-7 An example of a high-throughput mass spectrometry strategy commonly used for this purpose is a variation of the "shotgun" approach, involving in-solution digestion of a protein complex followed by onedimensional (1D) or two-dimensional (2D) liquid chromatography (LC) coupled with electrospray ionization (ESI) MS-MS.6-8 One of the applications of this method is for characterizing multiprotein complexes by identifying large numbers of proteins in a single data acquisition.9 Large-scale implementations of this strategy have been reported for yeast and Escherichia coli.10-12 To achieve a goal of characterizing large numbers of protein complexes13 isolated by affinity purification from Rhodopseudomonas palustris,14 an efficient protocol for digesting these complexes is required.
- 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:
- 931032
- Journal Information:
- Analytical Chemistry, Vol. 78, Issue 1; ISSN 0003-2700
- Country of Publication:
- United States
- Language:
- English
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