Direct Mass Spectrometric Analyses for Protein Chemistry Studies
For structural studies of various proteins, a combination of traditional sequence analysis and mass spectrometry (MS) has been effectively used in our laboratory. Protein sequencing methods and mass spectrometry are often plagued with the same problems in that samples are frequently contaminated with other materials. Many buffer components, salts, and solubilizing detergents can interfere or prevent the successful application of both methodologies. Membranes, made of materials such as polyvinyldifluoride (PVDF), have been invaluable in this regard for protein sequencing. These membranes have allowed for direct analysis of proteins from complex mixtures by allowing for separation by gel electrophoresis and subsequent electroblotting that immobilizes the protein and removes potentially interfering small molecules (Matsudaira, 1987). PVDF membranes are also stable to most organic solvents. Mass spectrometry is a sensitive bioanalytical method (McCloskey, 1990), but it is often difficult for the method to selectively discriminate against most species found in a sample, in search of the few components the scientist is truly interested. MS analysis of a sample containing a small amount of peptide in a great molar excess of buffer salts typically results in a mass spectrum mostly composed of buffer ions. Common desalting or chromatographic methods are often necessary prior to analysis by mass spectrometry, but this adds an additional step of complexity and increases the total analysis time.
KeywordsCysteine Polypeptide Trypsin MeOH Disulfide
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- Feng, R., Bell, A., Dumas, F., and Konishi, Y., 1990, Reduction/alkylation plus ionspray mass spectrometry: a fast and simple method for accurate counting of cysteines, disulfide bridges and free SH groups in proteins, Proc. 38th ASMS Conf. on Mass Spectrom. Allied Topics, Tucson, AZ: American Society for Mass Spectrometry: East Lansing, MI, pp 273–274.Google Scholar
- McCloskey, J. A. (Ed.)., 1990, Mass Spectrometry. San Diego, CA: Academic Press.Google Scholar