Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Reexamination Certificate
2002-02-06
2004-03-16
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
C436S173000, C530S402000, C530S412000
Reexamination Certificate
active
06706529
ABSTRACT:
BACKGROUND OF THE INVENTION
Historically, techniques such as Edman degradation have been extensively used for protein sequencing. See, Stark, in:
Methods in Enzymology,
25:103-120 (1972); Niall, in:
Methods in Enzymology,
27:942-1011 (1973); Gray, in:
Methods in Enzymology,
25:121-137 (1972); Schroeder, in:
Methods in Enzymology,
25:138-143 (1972); Creighton,
Proteins: Structures and Molecular Principles
(W. H. Freeman, N.Y., 1984); Niederwieser, in:
Methods in Enzymology,
25:60-99 (1972); and Thiede, et al.
FEBS-Lett.,
357:65-69 (1995). However, sequencing by collision-induced dissociation mass spectrometry (MS) methods (MS/MS sequencing) has rapidly evolved and has proved to be faster and require less protein than Edman techniques. See, Shevchenko, A., et al.,
Proc. Natl. Acad. Sci. (USA),
93:14440-14445 (1996); Wilm, et al.,
Nature,
379:466-469 (1996); Mark, J., “Protein structure and identification with MS/MS,” paper presented at the PE/Sciex Seminar Series, Protein Characterization and Proteomics: Automated high throughput technologies for drug discovery, Foster City, Calif. (March, 1998); and Bieman,
Methods in Enzymology,
193:455-479 (1990).
MS sequencing is accomplished either by using higher voltages in the ionization zone of the MS to randomly fragment a single peptide isolated from a protein digest, or more typically by tandem MS using collision-induced dissociation in the ion trap. See, Bieman, ibid. Several techniques can be used to select the peptide fragment used for MS/MS sequencing, including accumulation of the parent peptide fragment ion in the quadrapole MS unit (see, Mark, J. ibid.; Mann, M., paper presented at the IBC Proteomics conference, Boston, Mass. (Nov. 10-11, 1997); and Bieman,
Methods in Enzymology,
193:455479 (1990)), capillary electrophoretic separation coupled to ES-TOF MS detection (see, Aebersold, R. “Proteome analysis: Biological assay or data archive?,” paper presented at the IBC Proteomics conference, Coronado, Calif. (Jun. 11-12, 1998) and Smith, et al., in:
CRC Handbook of Capillary Electrophoresis: A Practical Approach
, Chp. 8, pgs 185-206 (CRC Press, Boca Raton, Fla., 1994)), or other liquid chromatographic separations (Niall, H. D., in:
Methods in Enzymology,
27:942-1011 (1973) and Creighton, T. E., Proteins: Structures and Molecular Principles (W. H. Freeman, N.Y., 1984)). The amino acid sequence of the peptide is deduced from the molecular weight differences observed in the resulting MS fragmentation pattern of the peptide using the published masses associated with individual amino acid residues in the MS (Biemann, K., in:
Methods in Enzymology.,
193:888 (1990), and has been codified into a semi-autonomous peptide sequencing algorithm (Hines, et al.,
J Am Soc Mass Spectrom,
3:326-336 (1992)).
For example, in the mass spectrum of a 1425.7 Da peptide (HSDAVFTDNYTR) isolated in an MS/MS experiment acquired in positive ion mode, the difference between the full peptide 1425.7 Da and the next largest mass fragment (y
11
, 1288.7 Da) is 137 Da. This corresponds to the expected mass of an N-terminal histidine residue that is cleaved at the amide bond. For this peptide, complete sequencing is possible as a result of the generation of high-abundance fragment ions that correspond to cleavage of the peptide at almost every residue along the peptide backbone. In the above-recited peptide sequence, the generation of an essentially complete set of positively-charged fragment ions that includes either end of the peptide is a result of the basicity of both the N- and C-terminal residues. When a basic residue is located at the N-terminus and/or C-terminus, most of the ions produced in the collision induced dissociation (CID) spectrum will contain that residue (see, Zaia, J., in:
Protein and Peptide Analysis by Mass Spectrometry
, J. R. Chapman, ed., pp. 29-41, Humane Press, Totowa, N.J. 1996; and Johnson, R. S., et al.,
Mass Spectrom. Ion Processes,
86:137-154 (1988)). since positive charge is generally localized at the basic site. The presence of a basic residue typically simplifies the resulting spectrum, since a basic site directs the fragmentation into a limited series of specific daughter ions. Peptides that lack basic residues tend to fragment into a more complex mixture of fragment ions that makes sequence determination more difficult.
Extending the concept of simplifying the CID spectrum of a peptide by including a charge concentrating moiety on either terminus of the peptide, others have demonstrated that attaching a hard positive charge to the N-terminus directs the production of a complete series of N-terminal fragment ions from a parent peptide in CID experiments regardless of the presence or absence of a basic residue at the N-terminus. See, Johnson, R. S., et al.,
Mass Spectrom. Ion Processes,
86:137-154 (1988); Vath, J. E., et al.,
Fresnius Zaia. Chem.,
331:248-252 (1988); Stults, J. T., et al.,
Anal. Chem.,
65:1703-1708 (1993); Zaia, J., et al.,
J. Am. Soc
. Mass Spectrom., 6:423-436 (1995); Wagner, D. S., et al.,
Biol. Mass Spectrom.,
20:419-425 (1991); and Huang, Z.-H., et al.,
Anal. Biochem.,
268:305-317 (1999). Theoretically, all fragment ions are produced by charge-remote fragmentation that is directed by the fixed-charged group. See, Tomer, K. B., et al.,
J. Am. Chem. Soc.,
105:5487-5488 (1983).
Peptides have been labeled with several classes of fixed-charge groups, including dimethylalkylammonium, substituted pyridinium, quaternary phosphonium, and sulfonium derivatives. Characteristics of useful labels include, ease of synthesis, increase in ionization efficiency of labeled peptides, and formation from a labeled peptide of a specific fragment ion series with minimal unfavorable label fragmentation. Zaia (in:
Protein and Peptide Analysis by Mass Spectrometry
, J. R. Chapman, ed., pp. 2941, Humana Press, Totowa, N.J., 1996) reported that the labels satisfying these criteria include those of the dimethylalkylammonium class and quarternary phosphonium derivatives. Moreover, it has been reported that substituted pyridinium derivatives are useful in high-energy CID. See, Bures, E. J., et al.,
Anal. Biochem.,
224:364-372 (1995) and Aebersold, R., et al., in:
Protein Science
, pp. 494-503 (Cambridge University Press, 1992).
Despite some progress in analytical methodology, protein identification remains a major bottleneck in field of proteomics. For example, it can require up to 18 hours to generate a protein sequence tag of sufficient length to allow the identification of a single purified protein from its predicted genomic sequence. Shevchenko, A., et al.,
Proc. Natl. Acad. Sci
. (
USA
), 93:14440-14445 (1996). Moreover, although unambiguous protein identification can be attained by generating a protein sequence tag (PST, see Clauser, K. R., et al.,
Proc. Natl. Acad. Sci
. (
USA
), 92:5072-5076 (1995) and Li, G., M., et al.,
Electrophoresis,
18:391-402 (1997)), limitations on the ionization efficiency of larger peptides and proteins restrict the intrinsic detection sensitivity of MS techniques and inhibit the use of MS for the identification of low abundance proteins. Furthermore, limitations on the mass accuracy of time of flight (TOF) detectors can also constrain the usefulness of presently utilized methods of MS/MS sequencing, requiring that proteins be digested by proteolytic and/or chemolytic means into more manageable peptides (see Ambler, R. P., in:
Methods in Enzymology,
25:143-154 (1972) and Gross, E., in:
Methods in Enzymol.,
11:238-255 (1967) prior to sequencing.
Two basic strategies have been proposed for the MS identification of proteins after their separation from a protein mixture: 1) mass profile fingerprinting (‘MS fingerprinting’) (see, James, P., et al.,
Biochem. Biophys. Res. Commun.,
195:58-64 (1993) and Yates, J. R., et al.,
Anal. Biochem.,
214:397-408 (1993)); and 2) sequencing of one or more peptide domains by MS/MS (‘MS/MS sequencing’)(see Mann, M., paper presented at the IBC Proteomics conference, Boston, Mass. (Nov. 10-11, 1997); Wilm, M.
Hall Michael P.
Peterson Jeffrey N.
Schneider Luke V.
Cole Monique T.
Target Discovery, Inc.
Townsend and Townsend / and Crew LLP
Warden Jill
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