Microsequencing, proteins

Microsequencers permit sequence analysis on minute amounts of protein. Microsequencing can be used in conjunction with two-dimensional electrophoretic separations of proteins such as that shown in Box 3-C. The proteins in the polyacrylamide gel are electropho-retically transferred onto a porous sheet (membrane) of an inert material such as polyvinyl difluoride.249-251 After staining, a selected spot is cut out and placed into the sequencer. To avoid the problems associated with blocked N termini, the protein may be treated with proteases on the membrane and the resulting peptide fragments may then be separated on a narrow-bore HPLC column and sequenced.240... 

LeGendre, N. and Matsudaira, P. 1988. Direct protein microsequencing from Immobilon-P transfer membrane. BioTechniques 6 154-159. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

Patton, W. F., Lam, L., Su, Q., Lui, M., Erdjument-Bromage, H., and Tempst, P. (1994) Metal chelates as reversible stains for detection of electroblotted proteins application to protein microsequencing and immunoblotting. Anal. Biochem. 220, 324-335. 

Posch, A., Chen, Z., Wheeler, C. et al. 1997. Characterization and identification of latex allergens by two-dimensional electrophoresis and protein microsequencing. J Allergy Clin Immunol 99 385-395. 

Protein microsequencing, peptide synthesis Site-directed mutagenesis Creation of specific mutant proteins Protein overproduction Appropriate host/vector systems... 

Wirth, P.J., Hoang, T.N., and Benjamin, T., 1995, Micropreparative immobilized pH gradient two-dimensional electrophoresis in combination with protein microsequencing for the analysis of human Uver proteins. Electrophoresis, 16 1946 - 1960. 

High performance capillary electrophoresis was introduced originally as an analytical tool. Now that instruments are equipped with automated fraction collection, however, capillary electrophoresis can be used for micropreparative collection of individual peaks separated from a mixture. Using the fraction collection feature, nanomolar amounts of solute such as proteins, peptides, oligonucleotides can be collected in amounts sufficient for microsequencing. An intersample washing procedure and use of well-formed capillaries aid in the prevention of artifacts.44... 

Peptide microsequencing has proved to be a powerful approach to identify protein components of mixtures,53-55,61 and has been applied to extracted proteins as a method to identify biological threats.56,83-85 Commercial and internet accessible search engines and databases provide adequate bioinformatics support to use sequence information produced by the mass spectrometer (e.g., http //www.matrixscience.com http //prospector.ucsf.edu/ ucsfhtml4.0/mstagfd.htm http //prowl.rockefeller.edu/). 

Muramoto K, Nokihara K, Ueda A, Kamiya H (1994) Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate. Biosci Biotechnol Biochem 58 300-304... 

Muramoto, K., Kamiya, H., and Kawauchi, H. (1984) The application of fluorescein isothiocyanate and high performance liquid chromatography for the microsequencing of proteins and peptides. Anal. Biochem. 141, 446. 

Braats JA, Mclntire KR (1978) In Catsimpoolas N (ed.) Electrophoresis 78, Elsevier, North Holland, New York Stone KL, Williams KR (1993) In Matsudaira P (ed.) A practical guide to protein and peptide ptuification for microsequencing, 2nd edn. Academic Press, San Diego, p 48... 

PI Kamp, R. M., Protein Structure Analysis -Preparation, Characterization and Microsequencing, Kamp, R. M. Choli-Papadopou-lou, Th. Wittmann-Liebold, B., Eds. Springer Heidelberg, (1997), p 231. 

Matsuidaira, P. T., ed. (1995) A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, San Diego, CA... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

Speicher, D.W. 1989. Microsequencing with PVDF membranes Efficient electroblotting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T. Hugli, ed.) pp. 24-35, Academic Press, San Diego. 

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