Identification of nucleotides, amino acids, or carbohydrates by

Details Identification of nucleotides, amino acids, or carbohydrates by Identification of nucleotides, amino acids, or carbohydrates by

1996-11-27

2000-01-25

Wortman, Donna

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

435 6, 436 87, 436 89, 436 94, 436173, 436175, 530412, C12Q 168, C12Q 170, G01N 2300

Patent

active

060176938

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

A number of approaches have been used in the past for applying the analytic power of mass spectrometry to peptides. Tandem mass spectrometry (MS/MS) techniques have been particularly useful. In tandem mass spectrometry, the peptide or other input (commonly obtained from a chromatography device) is applied to a first mass spectrometer which serves to select, from a mixture of peptides, a target peptide of a particular mass. The target peptide is then activated or fragmented to produce a mixture of the "target" or parent peptide and various component fragments, typically peptides of smaller mass. This mixture is then transmitted to a second mass spectrometer which records a fragment spectrum. This fragment spectrum will typically be expressed in the form of a bar graph having a plurality of peaks, each peak indicating the mass-to-change ratio (m/z) of a detected fragment and having an intensity value.
Although the bare fragment spectrum can be of some interest, it is often desired to use the fragment spectrum to identify the peptide (or the parent protein) which resulted in the fragment mixture. Previous approaches have typically involved using the fragment spectrum as a basis for hypothesizing one or more candidate amino acid sequences. This procedure has typically involved human analysis by a skilled researcher, although at least one automated procedure has been described. John Yates, III, et al., "Computer Aided Interpretation of Low Energy MS/MS Mass Spectra of Peptides" Techniques In Protein Chemistry II (1991), pp. 477-485, incorporated herein by reference. The candidate sequences can then be compared with known amino acid sequences of various proteins in the protein sequence libraries.
The procedure which involves hypothesizing candidate amino acid sequences based on fragment spectra is useful in a number of contexts but also has certain difficulties. Interpretation of the fragment spectra so as to produce candidate amino acid sequences is time-consuming, often inaccurate, highly technical and in general can be performed only by a few laboratories with extensive experience in tandem mass spectrometry. Reliance on human interpretation often means that analysis is relatively slow and lacks strict objectivity. Approaches based on peptide mass mapping are limited to peptide masses derived from an intact homogenous protein generated by specific and known proteolytic cleavage and thus are not generally applicable to mixtures of proteins.
Accordingly, it would be useful to provide a system for correlating fragment spectra with known protein sequences while avoiding the delay and/or subjectivity involved in hypothesizing or deducing candidate amino acid sequences from the fragment spectra.


SUMMARY OF THE INVENTION

According to the present invention, known amino acid sequences, e.g., in a protein sequence library, are used to calculate or predict one or more candidate fragment spectra. The predicted fragment spectra are then compared with an experimentally-derived fragment spectrum to determine the best match or matches. Preferably, the parent peptide, from which the fragment spectrum was derived has a known mass. Sub-sequences of the various sequences in the protein sequence library are analyzed to identify those sub-sequences corresponding to a peptide whose mass is equal to (or within a given tolerance of) the mass of the parent peptide in the fragment spectrum. For each sub-sequence having the proper mass, a predicted fragment spectrum can be calculated, e.g., by calculating masses of various amino acid subsets of the candidate peptide. The result will be a plurality of candidate peptides, each with a predicted fragment spectrum. The predicted fragment spectra can then be compared with the fragment spectrum derived from the tandem mass spectrometer to identify one or more proteins having sub-sequences which are likely to be identical with the sequence of the peptide which resulted in the experimentally-derived fragment spectrum.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is

REFERENCES:
patent: 5240859 (1993-08-01), Aebersold
patent: 5538897 (1996-07-01), Yates, III et al.
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Hunt, et al., "Protein Sequencing by Tandem Mass Spectrometry" Proc. Natl. Acad. Sci. USA, 83:6233-6237 (1986).
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Powell and Heiftje, Anal. Chim. Acta., vol. 100, pp. 313-327 (1978).
Smith, et al., "Collisional Activation and Collision-Activated Dissociation of Large Multiply Charged Polypetides and Proteins Produced by Electrospray Ionization," J. Am. Soc. Mass. Spectrom., 1:53-65 (1990).
Yates, III, et al., "Peptide Mass Maps: A Highly Informative Approach to Protein Identification," Analytical Biochemistry, 24:2-12 (1993).
Yates, III, et al., "Computer Aided Interpretation of Low Energy MS/MS Mass Spectra of Peptides," Techniques in Protein Chemistry II, pp. 477-485 (1991).
"Index of the Protein Sequence Database of the International Association of Protein Sequence Databanks (PIR-International)", Protein Seq. Data Anal., 5:67-192 (1993).

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