Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Patent
1992-04-30
1996-05-14
Snay, Jeffrey R.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
436180, 204450, 204451, 204452, G01N 2730, G01N 27447
Patent
active
055166989
DESCRIPTION:
BRIEF SUMMARY
This invention relates to methods and apparatus for carrying out chemical reactions between a plurality of reactants and in particular it is amenable to micro or nano scale operation and to the sequential chemical reactions required during such processes as construction or sequencing of proteins, oligonucleotides and polysaccharides.
A classic example of a sequential chemical reaction is the Edman protein sequencing technique involving the stepwise removal and identification of the N-terminal amino acid residues of a protein. The traditional Edman technique involves coupling the N-terminal amino acid residue to phenylisothiocyanate (PITC) in a solvent under alkaline or anhydrous conditions to form a phenylthiocarbamyl derivative. Excess PITC (usually at least 100 fold molar excess) is removed, typically by liquid extraction and the solvent also removed. The N-terminal amino acid, coupled to the PITC is subjected to cleavage by anhydrous acid to form an anilinothiazolinone (ATZ) derivative of the amino acid. The ATZ derivative is removed for subsequent chromatographic identification of the amino acid portion. The original protein is thus truncated, at its N-terminal by one amino acid allowing access to the formerly penultimate amino acid to be coupled, cleaved and identified with a subsequent Edman cycle. Further cycles can be undertaken to determine the entire sequence of the protein.
Some of the disadvantages of the traditional liquid phase Edman technique include the necessity to introduce plural incompatible reagents to achieve the coupling and cleavage steps for each cycle. In particular it is noted that strong alkali conditions are required in the coupling step whereas a strong acid environment achieves the cleavage. Clearly differentiation of these strong reagents is required to ensure a consistent removal of all the N-terminal residues without also generating spurious derivatives or uncoupling further non terminal residues. Furthermore the requirement for removal of the various volatile and non volatile auxiliary reagents and solvents during each cycle leads to sample loss and/or the formation of insoluble by-products. Relatively large volumes of sample and reagents are also required.
Effective automation of the Edman technique is first described in Edman and Begg, "A Protein Sequenator", in the European J. Biochem. 1, (1967), 80-91 and in U.S. Pat. No. 3,725,010. In these sequenators a liquid phase Edman technique is carried out in a thin film formed on the inside wall of a rotating reaction cell, now termed a "spinning cup". The spinning cup is located within a closed reaction chamber to maintain an inert atmosphere. Reagents are added to the cup by a system of pumps and valves and material removed by overflow over the lip of the spinning cup, vacuum evaporation or by dissolving or extracting in non polar solvents. The liquid reagents and solvents themselves form films on the walls of the spinning cup which pass over and interact with the sample film as the cup spins. The reagents dissolve the sample film and perform the coupling and cleavage stages of the Edman process, after which volatiles are removed by evacuation and the remaining sample film solvent extracted to transfer resulting amino acid thiazolone for identification.
It will be apparent that this dynamic system, requiring fluid and vacuum seals is difficult to construct and operate. The agitation induced by the spinning cup can cause the sample film to be overly extracted during washing or dislodged, in particular if the small protein being sequenced is a polypeptide. The initial protein sample must therefore be relatively large in volume and chain length. The drying of the protein onto the inner wall of the spinning cup must also be performed very carefully and slowly to achieve an even thin film while avoiding boiling and splatter during desiccation. Precise metering of reagents and solvents into the cup is also required to ensure consistency between cycles.
Laursen, in the European J. Biochem. 20 (1971), and Waschter et al., FEBS LeH
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Begg Geoffrey S.
Burgess Antony W.
Simpson Richard J.
Ludwig Institute for Cancer Research
Snay Jeffrey R.
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