Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Reexamination Certificate
1999-03-22
2001-08-21
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
C436S086000, C436S808000, C422S068100, C422S083000, C422S088000
Reexamination Certificate
active
06277644
ABSTRACT:
BACKGROUND
The invention relates generally to protein characterization and identification, and more specifically to an improved method and apparatus for determining the amino acid composition of a portion of a protein.
This and a related process and processing assembly are described in co-pending patent applications entitled “Protein Sequencing and Identification System”, Ser. No. 09/274,036, by Vincent Robert Farnsworth and Paul K. Cartier and “Multiple-Sample Cartridge Assembly for Automated Process”, Ser. No. 09/274,559, by Vincent Robert Farnsworth, both of which applications are filed on the same day as the present application, and both of which are incorporated herein by reference.
The identification and characterization of proteins is important to correlate particular proteins with disease states and to understand the biological function of proteins. Moreover, with the Human Genome project, there is an increasing need to link newly identified genes with their functional counterparts. Various methods have been described to do this, including, for example, digesting the protein with a specific enzyme such as trypsin and then use a mass spectrometer to analyze the resulting peptide mixture. The protein is identified by comparing the experimental mass profiles, including peptide fragmentation patterns, to the predicted profiles of known proteins stored in the databases.
An alternative approach to identifying a particular protein is two-dimensional polyacrylamide gel electrophoresis. Proteins from the 2D PAGE gel are analyzed, for example by Edman degradation. The Edman degradation process involves the sequential degradation of the N-terminus of a protein or polypeptide which comprises three basic stages: coupling, cleavage, and conversion. See Edman and Begg, “A Protein Sequenator”, European Journal of Biochem. 1 (1967) 80-91. The amino acids released from the Edman degradation reactions are then analyzed to determine a partial amino acid sequence, the results of which are correlated with sequence data in the existing DNA and protein databases. Unfortunately, identification of proteins by Edman degradation based sequencing is slow due to the serial nature of the process, which requires each cleaved amino acid to be analyzed prior to proceeding with the next.
These methods are of limited utility, in part because they are slow and expensive. Moreover, only one sample at a time is normally analyzed. What is needed is a way to identify proteins more rapidly and allow multiple samples to be processed in parallel.
SUMMARY
The present invention provides a system that satisfies the need, by providing a method and apparatus for determining the amino acid composition of at least a portion of at least one protein and using that information to identify the protein from a database of known sequences. The method involves coupling at least one protein with a coupling agent at one terminus of the protein so that a derivative of the amino acid at the terminus is formed, cleaving the coupled terminal amino acid from the protein such that a new amino acid is exposed at the terminus, repeating these steps of coupling and cleavage at least two times before simultaneously extracting the cleaved amino acids, identifying the extracted amino acids, determining the amino acid composition of at least a portion of the protein, and identifying the protein by comparing the amino acid composition against the amino acid compositions of a portion of known proteins.
One embodiment of the invention is a modification of the Edman degradation process which allows multiple cycles of coupling and cleavage to be performed without the normally required intervening steps of amino acid extraction and analysis. The invention enables the Edman degradation chemistry to be separated from amino acid analysis, and allows multiple cycles of coupling and cleavage to be performed prior to amino acid extractions. Preferably, the coupling and cleavage cycles are performed three or four times before the amino acids are extracted, converted, and subjected to compositional analysis. The amino acid composition information is then used to search a database of known protein or DNA sequences to identify the sample protein.
An apparatus for performing this method comprises a sample holder for holding the sample, a coupling agent supplier for supplying at least one coupling agent, a cleavage agent supplier for supplying a cleavage agent, a controller for directing the sequential supply of the coupling agents, cleavage agents, and other reagents necessary for performing the modified Edman degradation reactions, and an analyzer for analyzing amino acids.
REFERENCES:
patent: 3645689 (1972-02-01), Sjoquist
patent: 3717436 (1973-02-01), Penhasi et al.
patent: 3725010 (1973-04-01), Penhasi
patent: 3892531 (1975-07-01), Gilbert
patent: 4065412 (1977-12-01), Dreyer
patent: 5059540 (1991-10-01), Bailey
patent: 5082788 (1992-01-01), Farnsworth et al.
patent: 5156809 (1992-10-01), Hupe et al.
patent: 5185266 (1993-02-01), Boyd et al.
patent: 5223435 (1993-06-01), Kohr
patent: 5270213 (1993-12-01), Farnsworth
patent: 5439650 (1995-08-01), Tsugita et al.
patent: 5674743 (1997-10-01), Ulmer
A. A. Gooley, et al., A Role for Edman Degradation in Proteome Studies, Electrophoresis, 18, 1068-1072 (1997).
M. R. Wilkins, et al., From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Amino Acid Analysis, Bio/Technology, 14, 61-65 (1996).
V. Farnsworth et al., The Generation of Phenylthiocarbamyl or Anilinothiazolinone Amino Acids from the Postcleavage Products of the Edman Degradation, Analytical Biochemistry 215, 200-210 (1993).
P. Edman et al., A Protein Sequenator, European J. Biochem. 1, 80-91 (1967).
S. Katsui, et al., Short Communications, Biochem. J. (9165) 97, 25-26c.
Glycosite Protein Sequencer Operations Manual, p. 1-11.
Cartier, III Paul K.
Farnsworth Vincent R.
Beckman Coulter Inc.
Cole Monique T.
Grant Arnold
May William H.
Warden Jill
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