Proteolysis in Mixed Organic−Aqueous Solvent Systems: Applications for Peptide Mass Mapping Using Mass Spectrometry
- 6 April 2001
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 73 (11), 2682-2685
- https://doi.org/10.1021/ac001332p
Abstract
The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in <5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic−aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic−aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.Keywords
This publication has 26 references indexed in Scilit:
- The compact and expanded denatured conformations of apomyoglobin in the methanol‐water solventProtein Science, 1999
- Analysis of Reverse Phase Chromatographic SeparatedBacillus subtilisMembrane ProteinsAnalytical Letters, 1998
- Evaluation of the variables that affect resolution in delayed extraction MALDI-TOFInternational Journal of Mass Spectrometry and Ion Processes, 1997
- Probing the conformational state of apomyoglobin by limited proteolysisJournal of Molecular Biology, 1997
- Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cellsElectrophoresis, 1997
- The Methanol-induced Globular and Expanded Denatured States of Cytochrome : A Study by CD Fluorescence, NMR and Small-angle X-ray ScatteringJournal of Molecular Biology, 1996
- On Protein Denaturation in Aqueous−Organic Mixtures but Not in Pure Organic SolventsJournal of the American Chemical Society, 1996
- EmrE, an Escherichia coli 12-kDa Multidrug Transporter, Exchanges Toxic Cations and H+ and Is Soluble in Organic SolventsJournal of Biological Chemistry, 1995
- Improved Resolution and Very High Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast EvaporationAnalytical Chemistry, 1994
- Substrate specificity of enzymes in organic solvents vs. water is reversedJournal of the American Chemical Society, 1986