Microsequencing

Overall, the current literature about protein biochips has a tendency to make you sad highflying wishes, great plans, and a heap of petty problems that cannot be impressed by wishes. The crux seems to be in the production of aptamers that get at least as close to covering a similar variety of epitopes as antibodies do. Would that succeed with RNA and a number of unusual nucleotides or with RNA-peptide hybrids I have my doubts. Would nature not have already used something similar Are there organisms with aptamer immune systems  

Hamaguchi, N., et al. (2001). Aptamer Beacons for the Direct Detection of Proteins, Anal. Biochem. 294 126-131. 

Morris, K., et al. (1998). High Affinity Ligands from In Vitro Selection Complex Targets, Proc. Natl. Acad. Set USA 95 2902-2907. 

For microsequencing of a protein, you must perform several preparation steps. The protein has to be purified and prepared, and for the most important methods the protein must have a free N-terminus (or you have to cleave the protein into peptides). 

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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. 

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... 

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... 

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. 

Bhown, A.S., J.E. Mole, and J.C. Bennett, improved procedure for high-sensitivity microsequencing use of aminoethyl amino-propyl glass beads in the Beckman sequencer and the ultrasphere ODS column for PTH amino acid identification. Anal Biochem, 1981.110(2) 355-9. 

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... 

To test whether a polypeptide or other compound carried on a given bead has a derived biological activity, such as the ability to inhibit a certain enzyme, various assays that require only one bead can be devised. However, if a particular bead carries a compound of interest, how can it be identified The bead carries only a small amount of compound but it may be possible using microsequencing procedures to identify it. An alternative procedure is to use an encoding method to identify the beads. 

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). 

The purified glycoprotein can be used not only for amino acid microsequencing, but also for the determination of the structure of oligosaccharide chains. 

Nitrocellulose (e.g, Schleicher and Schull [Keene, NH] BA85) is a relatively inexpensive, if rather fragile, medium for blots, but PVDF membranes (e g, Problott, Applied Biosystems-Perkin Elmer, Foster City, CA) are needed for automated microsequencing of proteolytic fragments... 

Acrylamide (20%) with 0.5% bisacrylamide is appropriate for the separating gel. PVDF blots are stained with amido black, and all clearly visible bands are excised for direct microsequencing on the membrane, using an Applied Biosystems 476A or 494 sequencer (37)... 

Urea should not be used because its effects on amino groups interfere with the Edman degradation in subsequent microsequencing. For similar reasons, microsequencing can rarely be applied to chemical cleavage fragments. 

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

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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