Method for de novo peptide sequence determination

Details Method for de novo peptide sequence determination Method for de novo peptide sequence determination

1997-06-18

2003-06-24

Wang, Andrew (Department: 1639)

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Amino acid or sequencing procedure

C436S090000, C250S282000, C435S091500

Reexamination Certificate

active

06582965

ABSTRACT:

This application claims the priority benefit under 35 U.S.C. §119(a)-(d) of Great Britain Patent Application No. GB 9710582.9, filed on May 22, 1997.
FIELD OF THE INVENTION
The invention relates to a method for the determination of the precise linear sequence of amino acids in a peptide, polypeptide, or protein without recourse or reference to either a known pre-defined data base or to sequential amino acid residue analysis. As such, the method of the invention is a true, de novo peptide sequence determination method.
BACKGROUND OF THE INVENTION
The composition of a peptide, polypeptide, or protein as a sequence of amino acids is well understood. Each peptide, polypeptide, and protein is uniquely defined by a precise linear sequence of amino acids. Knowledge of the precise linear arrangement or sequence of amino acids in a peptide, polypeptide, or protein is required for various purposes, including DNA cloning in which the sequence of amino acids provides information required for oligonucleotide probes and polymerase chain reaction (“PCR”) primers. Knowledge of the exact sequence also allows the synthesis of peptides for antibody production, provides identification of peptides, polypeptides, and proteins, aids in the characterization of recombinant products, and is useful in the study of post-translational modifications.
A variety of sequencing methods are available to obtain the amino acid sequence information. For example, a series of chemical reactions, e.g., Edman reactions, or enzymatic reactions, e.g., exo-peptidase reactions, are used to prepare sequential fragments of the unknown peptide. Either an analysis of the sequential fragments or a sequential analysis of the removed amino acids is used to determine the linear amino acid sequence of the unknown peptide. Typically, the Edman degradation chemistry is used in modern automated protein sequencers.
In the Edman degradation, a peptide, polypeptide, or protein is sequenced by degradation from the N-terminus using the Edman reagent, phenylisothiocyanate (“PITC”). The degradation process involves three steps, i.e., coupling, cleavage, and conversion. In the coupling step, PITC modifies the N-terminal residue of the peptide, polypeptide, or protein. An acid cleavage then cleaves the N-terminal amino acid in the form of an unstable anilinothiazolinone (“ATZ”) derivative, and leaves the peptide, polypeptide, or protein with a reactive N-terminus and shortened by one amino acid. The ATZ derivative is converted to a stable phenylthiohydantoin in the conversion step for identification, typically with reverse phase high performance liquid chromatography (“RP-HPLC”). The shortened peptide, polypeptide, or protein is left with a free N-terminus that can undergo another cycle of the degradation reaction. Repetition of the cycle results in the sequential identification of each amino acid in the peptide, polypeptide, or protein. Because of the sequential nature of amino acid release, only one molecular substance can be sequenced at a time. Therefore, peptide, polypeptide, or protein samples must be extremely pure for accurate and efficient sequencing. Typically, samples must be purified with HPLC or SDS-PAGE techniques.
Although many peptide, polypeptide, and protein sequences have been determined by Edman degradation, currently, most peptide, polypeptide, and protein sequences are deduced from DNA sequences determined from the corresponding gene or cDNA. However, the determination of a protein sequence using a DNA sequencing technique requires knowledge of the specific nucleotide sequence used to synthesize the protein. DNA sequencing cannot be used where the nature of the protein or the specific DNA sequence used to synthesize the protein is unknown.
A peptide, polypeptide, or protein sequence may also be determined from experimental fragmentation spectra of the unknown peptide, polypeptide, or protein, typically obtained using activation or collision-induced fragmentation in a mass spectrometer. Tandem mass spectrometry (“MS/MS”) techniques have been particularly useful. In MS/MS, a peptide is first purified, and then injected into a first mass spectrometer. This first mass spectrometer serves as a selection device, and selects a target peptide of a particular molecular mass from a mixture of peptides and polypeptides or proteins, and eliminates most contaminants from the analysis. The target molecule is then activated or fragmented to form a mixture from the target or parent peptide of various peptides of a lower mass that are fragments of the parent. The mixture is then selected through a second mass spectrometer (i.e. step), generating a fragment spectrum.
Typically, in the past, the analysis of fragmentation spectra to determine peptide sequences has involved hypothesizing one or more amino acid sequences based on the fragmentation spectrum. In certain favorable cases, an expert researcher can interpret the fragmentation spectra to determine the linear amino acid sequence of an unknown peptide. The candidate sequences may then be compared with known amino acid sequences in protein sequence libraries.
In one strategy, the mass of each amino acid is subtracted from the molecular mass of the parent peptide to determine the possible molecular mass of a fragment, assuming that each amino acid is in a terminal position. The experimental fragment spectrum is then examined to determine if a fragment with such a mass is present. A score is generated for each amino acid, and the scores are sorted to generate a list of partial sequences for the next subtraction cycle. The subtraction cycle is repeated until subtraction of the mass of an amino acid leaves a difference of between −0.5 and 0.5, resulting in one or more candidate amino acid sequences. The highest scoring candidate sequences are then compared to sequences in a library of known protein sequences in an attempt to identify a protein having a sub-sequence similar or identical to the candidate sequence that generated the fragment spectrum.
Although useful in certain contexts, there are difficulties related to hypothesizing candidate amino acid sequences based on fragmentation spectra. The interpretation of fragmentation spectra is time consuming, can generally be performed only in a few laboratories that have extensive experience with mass spectrometry, and is highly technical and often inaccurate. Human interpretation is relatively slow, and may be highly subjective. Moreover, methods based on peptide mass mapping are limited to peptide masses derived from an intact homogeneous peptide, polypeptide, or protein generated by specific, known proteolytic cleavage, and, thus, are not applicable in general to a mixture of peptides, polypeptides, or proteins.
U.S. Pat. No. 5,538,897 to Yates, III et al. provides a method of correlating the fragmentation spectrum of an unknown peptide with theoretical spectra calculated from described peptide sequences stored in a database to match the amino acid sequence of the unknown peptide to that of a described peptide. 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 the experimentally-obtained fragment spectrum of the unknown protein to determine the best match or matches. Preferably, the mass of the unknown peptide is known. Sub-sequences of the various sequences in the protein sequence library are analyzed to identify those sub-sequences corresponding to a peptide having a mass equal to or within a given tolerance of the mass of the parent peptide in the fragmentation spectrum. For each sub-sequence having the proper mass, a predicted fragment spectrum can be calculated by calculating masses of various amino acid subsets of the candidate peptide. As a result, a plurality of candidate peptides, each having predicted fragment spectrum, is obtained. The predicted fragment spectra are then compared with the fragment spectrum obtained experimentally for the unknown protein to identify one or more prote

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