Peptides microsequencing

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/). 

Peptide microsequencing also provides an alternative approach to characterizing intact microorganisms but, the application where its potential... 

Gartner, D., Keilholz, W. and Seitz, H. (1994) Purification, characterization and partial peptide microsequencing of progesterone 5fJ-reductase from shoot cultures of Digitalis purpurea. Eur.. Biochem., 225,1125-32. 

A miniaturized protein and peptide microsequencer consisting of either a fused silica capillary reactor or a microreactor made of Teflon is described. The performance of the miniaturized sequencer was evaluated by sequencing 33 and 27 picomoles of myoglobin that were covalently attached to Sequelon-DITC. The products generated by the sequencer were analyzed using capillary electrophoresis with thermo-optical absorbance detection. This CE system provides reproducible migration time (< 0.4% of RSD) and detection limits of less than 4 fmol. 

Identification based on combined protein masses and peptide microsequencing... 

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... 

It should be pointed out that FAB, MALDI, and ESI can be used to provide ions for peptide mass maps or for microsequencing and that any kind of ion analyzer can support searches based only on molecular masses. Fragment or sequence ions are provided by instruments that can both select precursor ions and record their fragmentation. Such mass spectrometers include ion traps, Fourier transform ion cyclotron resonance, tandem quadrupole, tandem magnetic sector, several configurations of time-of-flight (TOF) analyzers, and hybrid systems such as quadrupole-TOF and ion trap-TOF analyzers. 

Microsequencing many peptides in a digested mixture may be coupled with bioinformatics to reveal information on all the component species. 

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... 

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... 

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

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. 

COI.ITMNST7F. Analytical reversed phase columns for proteins and peptides range from 50 to 250 mm in length having 4 to 5mm i.d. Columns with 2mm i.d. have also been employed for peptide separations in microsequencing applications. The use of microbore columns with less than 2 mm i.d. is severely hampered by instrumental constraints due to small extra-column dead volumes and difficulties in obtaining accurate... 

With peptide and oligonucleotide libraries prepared by split synthesis, the structure of a bound ligand on an individual bead can be determined by microsequencing [ 17,18]. This approach has been applied to testing libraries where the bead bound compounds are evaluated for their ability to bind to monoclonal antibodies or other recognition macromolecules. In one such approach, fluorescence-based methods are used to select the most... 

It is evident that /3-LPT is not the sole source of endogenous opioid peptides found in the pituitary gland now that dynorphin, an active peptide isolated from this organ as early as 1975, has been characterized.(127) This peptide differs from the /3-endorphins (lower mol wt, more basic, more persistent effect in GPI assay, and resistant to CNBr) and its first 13 residues (established by a microsequencing technique) commence with Leu-enkephalin at the N-terminal. The absence of Met5 accounts for its insensitivity toward CNBr. The synthetic tridecapeptide (12, dynorphin 1-13) and dynorphin itself... 

The elucidation of the primary structures of proctolin (22) and AKH (2Q), signaled the beginning of the structural identification of insect neuropeptides. These relatively small peptides (pentapeptide proctolin and decapeptide AKH) remained the only known insect peptide sequences for a number of years until, in the early 1980 s, accumulated technical advances in peptide isolation and microsequencing techniques facilitated the analysis of insect peptides (8-12 ... 

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

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