Method for de novo peptide sequence determination

Abstract

The present invention relates to a method for generating a library of peptides based on a peptide of a predetermined molecular mass and to a method of determining the amino acid sequence of the peptide from the library. A set of amino acids that can be present in the peptide are defined, and an allowed library of all possible sequences of the amino acids in the set having a total mass equal to the predetermined molecular mass, allowing for water lost in forming peptide bonds and for protonation, is generated. The allowed library may be generated by first generating a set of all combinations of amino acids having a total mass equal to the predetermined molecular mass of the peptide and then calculating all linear permutations of all amino acids in each such combination. Generally, the molecular mass is determined using a mass spectrometer. A theoretical fragmentation spectrum for every amino acid sequence in the allowed set may also be calculated for use in determining the amino acid sequence of the peptide. The fragmentation spectra may be of any type known in the art, such as a mass spectrum or a protease or chemical fragmentation spectrum. The theoretical fragmentation spectra calculated for each member of the allowed peptide library may be compared to an experimental fragmentation spectrum for the unknown peptide to determine the amino acid sequence of the unknown peptide.

Description

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 proteins having sub-sequences that are likely to be identical to the sequence of peptides that resulted in the experimentally-derived fragment spectrum. However, this technique cannot be used to derive the sequence of unknown, novel proteins or peptides having no sequence or sub-sequence identity with those pre-described or contained in such databases, and, thus, is not a de novo sequencing method.

Therefore, there remains a need for a true de novo sequencing method of determining the amino acid sequence of a peptide using mass spectrometry.

SUMMARY OF THE INVENTION

The present invention is directed to a method for generating a library of peptides, wherein each peptide in the library has a molecular mass corresponding to the same predetermined molecular mass. Typically, the library of peptides is then used to determine the amino acid sequence of an unknown peptide having the predetermined molecular mass. Preferably, the predetermined molecular mass used to generate the library is the molecular mass of the unknown peptide. Most preferably, the molecular mass of the unknown peptide is determined prior to the generation of the library using a mass spectrometer, such as a time-of-flight mass spectrometer.

The library is synthetic, i.e., not pre-described, and is typically generated each time a peptide is analyzed, based on the predetermined molecular mass of the unknown peptide. The library is generated by defining a set of all allowed combinations of amino acids that can be present in the unknown peptide, where the molecular mass of each combination corresponds to the predetermined molecular mass within the experimental accuracy of the device used to determine the molecular mass, allowing for water lost in peptide bond formation and for protonation, and generating an allowed library of all possible permutations of the linear sequence of amino acids in each combination in the set.

Generally, the present invention is directed to a method for determining the amino acid sequence of an unknown peptide, which comprises determining a molecular mass and an experimental fragmentation spectrum for the unknown peptide, comparing the experimental fragmentation spectrum of the unknown peptide to theoretical fragmentation spectra calculated for each individual member of an allowed synthetic peptide library, where the allowed peptide library is of the type described above, and identifying a peptide in the peptide library having a theoretical fragmentation spectrum that matches most closely the fragmentation spectrum of the unknown peptide, from which it is inferred that the amino acid sequence of the identified peptide in the allowed library represents the amino acid sequence of the unknown peptide.

The molecular mass for the unknown peptide may be determined by any means known in the art, but is preferably determined with a mass spectrometer. Allowed combinations of amino acids are chosen from a set of allowed amino acids that typically comprises the natural amino acids, i.e., tryptophan, arginine, histidine, glutamic acid, glutamine, aspartic acid, leucine, threonine, proline, alanine, tyrosine, phenylalanine, methionine, lysine, asparagine, isoleucine, cysteine, valine, serine, and glycine, but may also include other amino acids, including, but not limited to, non-natural amino acids and chemically modified derivatives of the natural amino acids, e.g., carbamidocysteine and deoxymethionine. Allowed combinations of amino acids are then calculated using one or more individual members of this set of amino acids, allowing for known mass changes associated with peptide bond formation, such that the total mass of each allowed combination corresponds to the predetermined mass of the unknown peptide to within the experimental accuracy to which this molecular mass of the unknown peptide was calculated, typically about 30 ppm. The set of allowed combinations is most easily calculated using an appropriately programmed computer. The allowed peptide library is assembled by permutation in all possible linear combinations of each allowed amino acid composition, and is also most easily constructed using an appropriately programmed computer. It should be noted that the term “allowed” with respect to amino acid combinations and libraries of peptides refers to combinations and libraries specific to the unknown peptide under investigation. The peptide library is constructed from the amino acid combinations, which in turn are calculated from the experimentally determined molecular mass. As unknown peptides of different mass are investigated, so different combinations of amino acids are allowed, and hence each unknown peptide of unique molecular mass gives rise to a unique peptide library.

The nature of the fragmentation process from which the theoretical fragmentation spectrum is calculated for every peptide in the allowed library may be of any type known in the art, such as a mass spectrum or a protease or chemical fragmentation spectrum. Preferably, both the molecular mass and the fragmentation spectrum for the unknown peptide are obtained from a tandem mass spectrometer. The immonium ion region of the mass spectrum used to determine the molecular mass may also be used to identify amino acids contained in the unknown peptide. The identity of these amino acids is then used to constrain the allowed library. The amino acid sequence of the peptide from the allowed library of peptides, having a calculated fragmentation spectrum that best fits the experimental fragmentation spectrum of the unknown peptide, corresponds to the amino acid sequence of the unknown peptide.

Although not required, the experimental fragmentation spectrum is generally normalized. A factor that is an indication of closeness-of-fit between the experimental fragmentation spectrum of the unknown peptide, polypeptide, or protein and each of the theoretical fragmentation spectra calculated for the peptide library may then be calculated to determine which of the theoretical fragmentation spectra best fits the experimental fragmentation spectrum. Preferably, peak values in the fragmentation spectra having an intensity greater than a predetermined threshold value are selected when calculating the indication of closeness-of-fit. The theoretical fragmentation spectrum that best fits the experimental fragmentation spectrum corresponds to the amino acid sequence in the allowed library that matches that of the unknown peptide, polypeptide, or protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the method of the invention.

FIG. 2 is a flow chart of a preferred embodiment of the invention.

FIG. 3 is the experimental mass spectrum used to determine the molecular mass of unknown Peptide X.

FIG. 4 is the immonium ion region of the mass spectrum shown in FIG. 3 , and identifies amino acids contained in unknown Peptide X.

FIG. 5 is the experimental fragmentation mass spectrum of Peptide X.

FIG. 6 is the experimental mass spectrum used to determine the molecular mass of Peptide Y.

FIG. 7 is the immonium ion region of the mass spectrum shown in FIG. 6 , and identifies amino acids contained in Peptide Y.

FIG. 8 is the experimental tandem mass spectrum of Peptide Y.

FIG. 9 is the experimental mass spectrum used to determine the molecular mass of Peptide Z.

FIG. 10 is the immonium ion region of the mass spectrum shown in FIG. 9 , and identifies amino acids contained in Peptide Z.

FIG. 11 is the experimental tandem mass spectrum of Peptide Z.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a de novo method for determining the sequence of an unknown peptide without reference to any experimentally determined peptide or nucleotide sequence, and without recourse to a sequential and step-wise identification and ordering of individual amino acid residues, such as the Edman degradation process or interpretation of conventional mass spectrometry fragmentation patterns. In the method of the invention, a library of theoretical peptide sequences is generated from a predetermined molecular mass, preferably the experimentally determined molecular mass of an unknown peptide. As such, this library must contain the amino acid sequence of the unknown peptide, as well as that of any other peptide having the predetermined molecular mass. The precise amino acid sequence of the unknown is identified by applying standard correlation functions to select that peptide from the synthetic library whose calculated, i.e., theoretical, fragmentation spectrum most closely matches the fragmentation pattern of the unknown. In the preferred embodiment, the fragmentation spectrum is a mass spectrum and the correlation method is the function described in U.S. Pat. No. 5,538,897, the contents of which are incorporated herein in their entirety by reference. Preferably, the theoretical fragmentation spectra are generated and matched to the fragmentation pattern of the unknown using an appropriately programmed computer.

The invention may be better understood by reference to the flow chart provided in FIG. 1 . Where the peptide is a protein or large polypeptide, the protein or large polypeptide may be cleaved to form a peptide pool by means well known in the art. The unknown peptide (“Peptide X”) is then separated from the pool by HPLC or any other means known in the art, preferably mass spectrometry, and the molecular mass of Peptide X is determined. Although there are a number of methods for determining the molecular mass of Peptide X the preferred method is again mass spectrometry.

A set of amino acids that theory or experimental results teach may be included in Peptide X is then defined for consideration in determining the sequence of Peptide X. The defined set of amino acids may include modified or unnatural amino acids in addition to natural amino acids.

Typically, the method of the invention requires a “naked” peptide when determining the amino acid sequence. Therefore, the peptide should be free of any individual amino acids that are covalently modified by post-translational modification, such as, e.g., glycosylation, which involves the attachment of carbohydrate to the side chain of certain amino acids. Where the method of the invention is used to determine the amino acid sequence of a post-translationally modified peptide, the modifications are typically removed from the peptide prior to the analysis, taking due care to leave the peptide intact. Methods for removing post-translational modifications from peptides are well known in the art, and include, for example, the removal of N-linked carbohydrates with enzymes, such as peptide-N-glycosidase F (PNGase F), endo-glycosidases, mixtures of exo-glycosidases, etc., and the removal of phosphate modification with phosphatases. In addition, other techniques for removing modifications occasionally found on peptides are well known in the art. However, where a specific modification to a specific amino acid is known to be present in the unknown peptide, the modified amino acid may be included in the defined set of amino acids that theory or experimental results teach may be included in Peptide X, and, thus, the sequence of the peptide containing the modified peptide may be determined with the method of the present invention.

All combinations of amino acids having a total mass equal to the measured mass of Peptide X are calculated, allowing for water lost in determining peptide links, protonation, etc. Any individual amino acid may be included as part of any given combination at any integral stoichiometry up to the amount consistent with the mass determined for Peptide X. These combinations comprise all of the allowed combinations of amino acids combinations for Peptide X, and, therefore, the actual amino acid compositions of Peptide X will be represented in one and only one of these combinations. Furthermore, these combinations are generally peptide specific.

An allowed library of linear peptides is then constructed from the allowed combinations of amino acids. The allowed library is constructed by generating all possible linear permutations of the sequence of amino acids in each combination, using all the amino acids in each combination. The allowed library comprises all such permutations of the amino acids, and therefore must include Peptide X. The allowed library of peptides having the same molecular mass as Peptide X is typically constructed independently and ab initio for each new unknown peptide that is sequenced. That is, a new library is typically constructed as part of each analysis, and for only that analysis. However, as will be clear to one of ordinary skill in the art, once a library of all peptides, polypeptides, or proteins having a given molecular mass has been constructed, that library may be used for the determination of the amino acid sequence of any other peptide, polypeptide, or protein of that particular molecular mass.

This differs fundamentally from existing data base approaches in which a single data base of known sequences, which is subject to periodic updates and refinements based on the availability of experimentally determined sequences, is used for all analyses. As a result, with the method of the present invention, the determination of new and previously unknown peptides sequences that are not present in any experimentally determined peptide sequence library is possible by direct peptide analysis in a non-step-wise, operator-independent automated process. In addition, the method of the invention is not constrained to the conventional twenty amino acids, or to their conventional modifications.

In a preferred embodiment, as shown in the flow chart provided in FIG. 2 , additional information relating to Peptide X is used to place constraints on the allowed combinations of amino acids and/or allowed peptide sequences in the library, and, thus, reduce the number of possible sequences. Useful information related to Peptide X includes, but is not limited to, partial amino acid composition. For example, the mass spectrum used to determine the mass of Peptide X may include fragments that can be used to identify specific amino acids present in Peptide X. Where it is known that certain amino acids are definitely present in Peptide X, constraints are placed on the allowed combinations and allowed library by requiring the identified amino acids to be present in all combinations and, thus, in every peptide present in the library.

Again with reference to FIGS. 1 and 2 , the allowed library, which has preferably been constrained, is then used as the basis for generating theoretical fragmentation patterns that are compared to the experimental fragmentation pattern obtained for Peptide X. The fragmentation patterns may be obtained by any suitable means known in the art. Preferably, the fragmentation patterns are mass spectra, and the method used to match the theoretical and experimental mass spectra is that disclosed in U.S. Pat. No. 5,538,897. However, protease or chemical fragmentation, coupled to HPLC separation of the fragments, may also be used to obtain the experimental fragmentation patterns.

Preferably, in a determination of the amino acid sequence of Peptide X, the molecular mass of Peptide X is determined with high accuracy, typically, to within about 30 ppm (parts per million). An example of such a spectrum is provided in FIG. 3 , where the molecular mass of Peptide X is determined from the peak at 774.3928 daltons. In addition, as a result of the partial fragmentation of Peptide X that can occur, fragments that identify certain amino acids that are contained in Peptide X are also observed, allowing the peptide library to be constrained. An example of this portion of the mass spectrum for Peptide X is provided in FIG. 4 .

Peptide X is then subjected to collision-induced dissociation in a mass spectrometer. The parent peptide and its fragments are then introduced into the second mass spectrometer that provides an intensity or count and the mass to charge ratio, m/z, for each of the fragments in the fragment mixture. Each fragment ion is represented in a bar graph in which the abscissa value is m/z and the ordinate value is the intensity. A variety of mass spectrometer types can be used, including, but not limited to a triple-quadrapole mass spectrometry, Fourier-transform cyclotron resonance mass spectrometry, tandem time-of-flight mass spectrometry, and quadrapole ion trap mass spectrometry.

The experimental fragment spectrum is then compared to the mass spectra predicted for the sequences of the allowed library to identify one or more predicted mass spectra that closely match the experimental mass spectrum. Because the allowed library includes all permutations of amino acid sequences that have a total mass corresponding to that of Peptide X, Peptide X must be represented in the allowed library.

The predicted fragmentation spectra may be obtained and compared to the experimental fragmentation spectrum by employing a method that involves first normalizing the experimental fragmentation spectrum. This may be accomplished by converting the experimental fragmentation spectrum to a list of masses and intensities. The peak values for Peptide X are removed, and the square root of the remaining intensity values is calculated, and normalized to a maximum value of 100. The 200 most intense ions are divided into ten mass regions, and the maximum intensity within each region is again normalized to 100. Each ion within 3.0 daltons of its neighbor on either side is given an intensity value equal to the greater of the intensity of the ion or that of its neighbor. Other normalization methods can be used, and it is possible to perform the analysis without normalizing. However, in general, normalization is preferred. In particular, maximum normalized values, the number of intense ions, the number of mass regions, and the size of the window for assuming the intensity value of a near neighbor may all be independently varied to larger or smaller values.

A fragment mass spectrum is predicted for each of the candidate sequences. The fragment mass spectrum is predicted by calculating the fragment ion masses for the type b and y ions for the amino acid sequence. When a peptide is fragmented and the charge is retained on the N-terminal cleavage fragment, the resulting ion is labelled as a b-type ion. If the charge is retained on the C-terminal fragment, it is labelled a y-type ion. Masses for type b ions were calculated by summing the amino acid masses and adding the mass of a proton. Masses for type y ions were calculated by summing, from the C-terminus, the masses of the amino acids and adding the mass of water and a proton to the initial amino acid. In this way, it is possible to calculate an m/z value for each fragment.

However, in order to provide a predicted mass spectrum, it is also necessary to assign an intensity value for each fragment. Although it is often possible to predict, on a theoretical basis, intensity value for each fragment, this procedure is difficult, and it has been found useful to assign intensities in the following fashion. The value of 50.0 is assigned to each b and y ion. To masses of 1 dalton on either side of the fragment ion, an intensity of 25.0 is assigned. Peak intensities of 10.0 are assigned at masse peaks 17.0 and 18.0 daltons below the m/z of each b and y ion location to account for both NH 3 and H 2 O loss, and peak intensities of 10.0 are assigned to mass peaks 28.0 daltons below each type b ion location to account for CO loss.

After calculation of predicted m/z values and assignment of intensities, it is preferred to calculate a measure of closeness-of-fit between the predicted mass spectra and the experimentally-derived fragment spectrum. A number of methods for calculating closeness-of-fit are available. For example, a two-step method may be used that includes calculating a preliminary closeness-of-fit score, referred to here as S p , and calculating a correlation function for the highest-scoring amino acid sequences. In the preferred embodiment, S p is calculated using the following formula:

S p =(Σ i m )* n* i (1+β)*(1−ρ)/ n τ   (1)

where i m are the matched intensities, n i are the number of matched fragment ions, β is the type b and y ion continuity, ρ is the presence of immonium ions and their respective amino acids in the predicted sequence, and n τ is the total number of fragment ions. The factor, β, evaluates the continuity of a fragment ion series. If there is a fragment ion match for the ion immediately preceding the current type b or y ion, β is incremented by 0.075 from an initial value of 0.0. This increases the preliminary score for those peptides matching a successive series of type b and y ions, since extended series of ions of the same type are often observed in MS/MS spectra. The factor ρ evaluates the presence of immonium ions in the low mass end of the mass spectrum.

The detection of immonium ions may be used diagnostically to determine the presence of certain types of amino acids in the sequence. For example, if immonium ions are present at 110.0, 120.0, or 136.0±1.0 daltons in the processed data file of the unknown peptide with normalized intensities greater than 40.0, indicating the presence of histidine, phenylalanine, and tyrosine respectively, then the sequence under evaluation is checked for the presence of the amino acid indicated by the immonium ion. The preliminary score, S p , for the peptide is either increased or decreased by a factor of 1−ρ, where ρ is the sum of the penalties for each of the three amino acids whose presence is indicated in the low mass region. Each individual ρ can take on the value of −0.15 if there is a corresponding low mass peak, and the amino acid is not present in the sequence, +0.15 if there is a corresponding low mass peak and the amino acid is present in the sequence, or 0.0 if the low mass peak is not present. The total penalty can range from −0.45, where all three low mass peaks are present in the spectrum, but are not present in the sequence, to +0.45, where all three low mass peaks are present in the spectrum, and are present in the sequence.

Following the calculation of the preliminary closeness-of-fit score S p . the predicted mass spectra having the highest S p scores are selected for further analysis using the correlation function. The number of candidate predicted mass spectra that are selected for further analysis will depend largely on the computational resources and time available.

For purposes of calculating the correlation function, the experimentally-derived fragment spectrum is typically preprocessed in a fashion somewhat different from preprocessing employed before calculating S p . For purposes of the correlation function, the precursor ion is removed from the spectrum, and the spectrum is divided into 10 sections. Ions in each section are then normalized to 50.0. The section-wise normalized spectra are then used for calculating the correlation function. The discrete correlation between the two functions may be calculated as: $$ R τ = ∑ i = 0 n - 1 ⁢ x i ⁢ y i + τ ( 2 )

where τ is a lag value. The discrete correlation theorem states that the discrete correlation of two real functions x and y is one member of the discrete Fourier transform pair

R τ ⇄X τ Y*τ   (3)

where X(t) and Y(t) are the discrete Fourier transforms of x(i) and y(i), and the Y* denotes complex conjugation. Therefore, the cross-correlations can be computed by Fourier transformation of the two data sets using the fast Fourier transform (FFT) algorithm, multiplication of one transform by the complex conjugate of the other, and inverse transformation of the resulting product.

The predicted spectra as well as the pre-processed unknown spectrum may be zero-padded to 4096 data points, since the MS/MS spectra are not periodic, as intended by the correlation theorem, and the FFT algorithm requires N to be a integer power of two, so the resulting end effects need to be considered. The final score attributed to each candidate peptide sequence is R(0) minus the mean of the cross-correlation function over the range −75<t<75. This modified “correlation parameter”, described in Powell and Heiftje, Anal. Chim. Acta, 100:313-327 (1978), shows better discrimination over just the spectral correlation coefficient R(0). The raw scores are normalized to 1.0. Preferably, the output includes the normalized raw score, the candidate peptide mass, the unnormalized correlation coefficient, the preliminary score, the fragment ion continuity β, the immonium ion factor τ, the number of type b and y ions matched out of the total number of fragment ions, their matched intensities, the protein accession number, and the candidate peptide sequence.

The correlation function can be used to automatically select one of the predicted mass spectra as corresponding to the experimentally-derived fragment spectrum. Preferably, however, a number of sequences from the library are output and final selection of a single sequence is done by a skilled operator.

Depending on the computing and time resources available, it may be advantageous to employ data-reduction techniques. Preferably these techniques will emphasize the most informative ions in the spectrum while not unduly affecting search speed. One technique involves considering only some of the fragment ions in the MS/MS spectrum, which, for a peptide may contain as many as 3,000 fragment ions. According to one data reduction strategy, the ions are ranked by intensity, and some fraction of the most intense ions is used for comparison. Another approach involves subdividing the spectrum into a small number of regions, e.g., about 5, and using the 50 most intense ions in each region as part of the data set. Yet another approach involves selecting ions based on the probability of those ions being sequence ions. For example, ions could be selected which exist in mass windows of 57 through 186 daltons, i.e., the range of mass increments for the 20 common amino acids from glycine to tryptophan that contain diagnostic features of type b or y ions, such as losses of 17 or 18 daltons, corresponding to ammonia and water, or a loss of 28 daltons, corresponding to CO.

A number of different scoring algorithms can be used for determining preliminary closeness of fit or correlation. In addition to scoring based on the number of matched ions multiplied by the sum of the intensity, scoring can be based on the percentage of continuous sequence coverage represented by the sequence ions in the spectrum. For example, a 10 residue peptide will potentially contain 9 each of b and y type sequence ions. If a set of ions extends from b 1 to b 9 , then a score of 100 is awarded, but if a discontinuity is observed in the middle of the sequence, such as missing the b 5 ion, a penalty is assessed. The maximum score is awarded for an amino acid sequence that contains a continuous ion series in both the b and y directions.

In the event the described scoring procedures do not delineate an answer, an additional technique for spectral comparison can be used in which the database is initially searched with a molecular weight value and a reduced set of fragment ions. Initial filtering of the database occurs by matching sequence ions and generating a score with one of the methods described above. The resulting set of answers will then undergo a more rigorous inspection process using a modified full MS/MS spectrum.

For the second stage analysis, one of several spectral matching approaches developed for spectral library searching is used. This will require generating a “library spectrum” for the peptide sequence based on the sequence ions predicted for that amino acid sequence. Intensity values for sequence ions of the “library spectrum” will be obtained from the experimental spectrum. If a fragment ion is predicted at m/z 256, then the intensity value for the ion in the experimental spectrum at m/z 256 will be used as the intensity of the ion in the predicted spectrum. Thus, if the predicted spectrum is identical to the “unknown” spectrum, it will represent an ideal spectrum. The spectra will then be compared using a correlation function. In general, it is believed that the majority of computational time for the above procedure is spent in the iterative search process. By multiplexing the analysis of multiple MS/MS spectra in one pass through the database, an overall improvement in efficiency will be realized. In addition, the mass tolerance used in the initial pre-filtering can affect search times by increasing or decreasing the number of sequences to analyze in subsequent steps.

Another approach to speed up searches involves a binary encryption scheme where the mass spectrum is encoded as peak/no peak at every mass depending on whether the peak is above a certain threshold value. If intensive use of a protein sequence library is contemplated, it may be possible to calculate and store predicted mass values of all sub-sequences within a predetermined range of masses so that at least some of the analysis can be performed by table look-up rather than calculation.

EXAMPLES

The following non-limiting examples are merely illustrative of the preferred embodiments of the present invention, and are not to be construed as limiting the invention, the scope of which is defined by the appended claims.

Example 1

The amino acid sequence of unknown Peptide X was determined using the method of the invention. The molecular mass of Peptide X was first determined using a matrix-assisted laser-desorption time-of-flight mass spectrometer (Voyager Elite, manufactured by Perseptive Biosystems) with delayed extraction and post source decay. As shown in FIG. 3 , the mass of the protonated form of peptide X is 774.3928 daltons, which indicates a sass of 773.3928 daltons for Peptide X.

The set of amino acids that are possibly part of Peptide X were then defined for consideration in the analysis. The defined set of amino acids with the molecular mass of each amino acid less the mass of the one water molecule lost during peptide bond formation is provided below. The molecular masses are given in daltons or a.m.u.

tryptophan =	186.079313	carbamido cysteine =	160.03065
arginine =	156.10111	phenylalanine =	147.068414
histidine =	137.058912	methionine =	131.04085
glutamic acid =	129.042593	lysine =	128.094963
glutamine =	128.058577	asparagine =	114.042927
aspartic acid =	115.026943	isoleucine =	113.084064
leucine =	113.084064	cysteine =	103.009185
threonine =	101.047678	valine =	99.068414
proline =	97.052764	serine =	87.032028
alanine =	71.037114	glycine =	57.021464
tyrosine =	163.063328

The allowed combinations of amino acids for Peptide X were determined by first determining the molecular mass of Peptide X, as described above, to an experimental accuracy of 30 ppm (parts per million). Therefore, each allowed combination of amino acids in the allowed library must have a total mass of 773.3928±30 ppm. In addition to providing the molecular mass of Peptide X, the first mass spectrum also confirmed the presence of certain amino acids in Peptide X. The immonium region of this mass spectrum, which shows the presence of these amino acids, is given in FIG. 4 . In particular, the immonium region of the spectrum indicates the presence of arginine with a characteristic mass of 174.988, leucine/isoleucine with a characteristic mass at 85.8851 (these amino acids have the same mass, and are therefore not distinguishable by mass alone), histidine with a characteristic mass at 109.823, and tyrosine with a characteristic mass at 135.915. Therefore, it was possible to constrain the allowed library to sets containing arginine, leucine/isoleucine, histidine, and tyrosine, having a total molecular mass of 773.3928±30 ppm.

To determine the sets of amino acids that have a total molecular mass of 773.3928±30 ppm, the following equation was applied:

MM x =Σ(histidine)+(tyrosine)+(leucine/isoleucine)+(arginine)+(H 2 O)+(aa 1 )+ - - - +(aa n ),

where aa 1 - - - aa n are any of the allowed amino acids, other than arginine, isoleucine, histidine, and tyrosine. The only combinations of amino acids that can have a total molecular mass of 773.3928±30 ppm are as follows:

1) tryptophan, arginine, leucine/isoleucine, histidine, and tyrosine.

2) glutamic acid, glycine, arginine, leucine/isoleucine, histidine, and tyrosine.

3) alanine, aspartic acid, arginine, leucine/isoleucine, histidine, and tyrosine.

These combinations constitute the allowed sets of amino acids for Peptide X.

In addition, Peptide X was obtained by a tryptic cleavage, and, therefore, from the accepted specificity of trypsin, Peptide X must also have lysine or arginine as its carboxy terminal amino acid. With this constraint, the allowed library of linear peptides was constructed from all individual linear permutations of combinations 1, 2, and 3. The allowed library includes 528 linear peptides, one set of 264 peptides containing isoleucine (SEQ ID NOS:1-264) (shown below) and a corresponding set of 264 peptides in which isoleucine is replaced by leucine (not shown).

1)   YIHWR
2)   IYHWR
3)   YHIWR
4)   HYIWR
5)   IHYWR
6)   HIYWR
7)   YIWHR
8)   IYWHR
9)   YWIHR
10)  WYIHR
11)  IWYHR
12)  WIYHR
13)  YHWIR
14)  HYWIR
15)  YWHIR
16)  WYHIR
17)  HWYIR
18)  WHYIR
19)  IHWYR
20)  HIWYR
21)  IWHYR
22)  WIHYR
23)  HWIYR
24)  WHIYR
25)  YIHEGR
26)  IYHEGR
27)  YHIEGR
28)  HYIEGR
29)  IHYEGR
30)  HIYEGR
31)  YIEHGR
32)  IYEHGR
33)  YEIHGR
34)  EYIHGR
35)  IEYHGR
36)  EIYHGR
37)  YHEIGR
38)  HYEIGR
39)  YEHIGR
40)  EYHIGR
41)  HEYIGR
42)  EHYIGR
43)  IHEYGR
44)  HIEYGR
45)  IEHYGR
46)  EIHYGR
47)  HEIYGR
48)  EHIYGR
49)  YIHGER
50)  IYHGER
51)  YHIGER
52)  HYIGER
53)  IHYGER
54)  HIYGER
55)  YIGHER
56)  IYGHER
57)  YGIHER
58)  GYIHER
59)  IGYHER
60)  GIYHER
61)  YHGIER
62)  HYGIER
63)  YGHIER
64)  GYHIER
65)  HGYIER
66)  GHYIER
67)  IHGYER
68)  HIGYER
69)  IGHYER
70)  GIHYER
71)  HGIYER
72)  GHIYER
73)  YIEGHR
74)  IYEGHR
75)  YEIGHR
76)  EYIGHR
77)  IEYGHR
78)  EIYGHR
79)  YIGEHR
80)  IYGEHR
81)  YGIEHR
82)  GYIEHR
83)  IGYEHR
84)  GIYEHR
85)  YEGIHR
86)  EYGIHR
87)  YGEIHR
88)  GYEIHR
89)  EGYIHR
90)  GEYIHR
91)  IEGYHR
92)  EIGYHR
93)  IGEYHR
94)  GIEYHR
95)  EGIYHR
96)  GEIYHR
97)  YHEGIR
98)  HYEGIR
99)  YEHGIR
100) EYHGIR
101) HEYGIR
102) EHYGIR
103) YHGEIR
104) HYGEIR
105) YGHEIR
106) GYHEIR
107) HGYEIR
108) GHYEIR
109) YEGHIR
110) EYGHIR
111) YGEHIR
112) GYEHIR
113) EGYHIR
114) GEYHIR
115) HEGYIR
116) EHGYIR
117) HGEYIR
118) GHEYIR
119) EGHYIR
120) GEHYIR
121) IHEGYR
122) HIEGYR
123) IEHGYR
124) EIHGYR
125) HEIGYR
126) EHIGYR
127) IHGEYR
128) HIGEYR
129) IGHEYR
130) GIHEYR
131) HGIEYR
132) GHIEYR
133) IEGHYR
134) EIGHYR
135) IGEHYR
136) GIEHYR
137) EGIHYR
138) GEIHYR
139) HEGIYR
140) EHGIYR
141) HGEIYR
142) GHEIYR
143) EGHIYR
144) GEHIYR
145) YIHDAR
146) IYHDAR
147) YHIDAR
148) HYIDAR
149) IHYDAR
150) HIYDAR
151) YIDHAR
152) IYDHAR
153) YDIHAR
154) DYIHAR
155) IDYHAR
156) DIYHAR
157) YHDIAR
158) HYDIAR
159) YDHIAR
160) DYHIAR
161) HDYIAR
162) DHYIAR
163) IHDYAR
164) HIDYAR
165) IDHYAR
166) DIHYAR
167) HDIYAR
168) DHIYAR
169) YIHADR
170) IYHADR
171) YHIADR
172) HYIADR
173) IHYADR
174) HIYADR
175) YIAHDR
176) IYAHDR
177) YAIHDR
178) AYIHDR
179) IAYHDR
180) AIYHDR
181) YHAIDR
182) HYAIDR
183) YAHIDR
184) AYHIDR
185) HAYIDR
186) AHYIDR
187) IHAYDR
188) HIAYDR
189) IAHYDR
190) AIHYDR
191) HAIYDR
192) AHIYDR
193) YIDAHR
194) IYDAHR
195) YDIAHR
196) DYIAHR
197) IDYAHR
198) DIYAHR
199) YIADHR
200) IYADHR
201) YAIDHR
202) AYIDHR
203) IAYDHR
204) AIYDHR
205) YDAIHR
206) DYAIHR
207) YADIHR
208) AYDIHR
209) DAYIHR
210) ADYIHR
211) IDAYHR
212) DIAYHR
213) IADYHR
214) AIDYHR
215) DAIYHR
216) ADIYHR
217) YHDAIR
218) HYDAIR
219) YDHAIR
220) DYHAIR
221) HDYAIR
222) DHYAIR
223) YHADIR
224) HYADIR
225) YAHDIR
226) AYHDIR
227) HAYDIR
228) AHYDIR
229) YDAHIR
230) DYAHIR
231) YADHIR
232) AYDHIR
233) DAYHIR
234) ADYHIR
235) HDAYIR
236) DHAYIR
237) HADYIR
238) AHDYIR
239) DAHYIR
240) ADHYIR
241) IHDAYR
242) HIDAYR
243) IDHAYR
244) DIHAYR
245) HDIAYR
246) DHIAYR
247) IHADYR
248) HIADYR
249) IAHDYR
250) AIHDYR
251) HAIDYR
252) AHIDYR
253) IDAHYR
254) DIAHYR
255) IADHYR
256) AIDHYR
257) DAIHYR
258) ADIHYR
259) HDAIYR
260) HADIYR
261) HADIYR
262) AHDIYR
263) DAHIYR
264) ADHIYR

The method of U.S. Pat. No. 5,538,897 was then used to match Peptide X to this library by MS/MS. The experimental tandem mass spectrum of Peptide X is shown in FIG. 5 , and the 10 top ranking peptides matched to this spectrum are provided below (SEQ ID NOS:228, 238, 227, 237, 186, 226, 192, 225, 219, and 217, respectively). It was determined that the sequence of Peptide X is that of the top ranked peptide, AHYDIR (SEQ ID NO:228).

Rank/	(M +
Sp	H)	Cn	C * 10 4	Sp	Ions	Reference	Peptide
1/1	774.9	1.0000	1.8118	491.0	11/15	p(228)	(−)AHYDIR
2/3	774.9	0.9308	1.6864	386.2	10/15	p(238)	(−)AHDYIR
3/2	774.9	0.8012	1.4516	414.3	10/15	p(227)	(−)HAYDIR
4/5	774.9	0.7319	1.3262	320.5	9/15	p(237)	(−)HADYIR
5/1	774.9	0.7168	1.2987	491.0	11/15	p(186)	(−)AHYIDR
6/12	774.9	0.6131	1.1108	248.3	9/15	p(226)	(−)AYHDIR
7/3	774.9	0.6033	1.0930	386.2	10/15	p(192)	(−)AHIYDR
8/9	774.9	0.5878	1.0651	264.1	9/15	p(225)	(−)YAHDIR
9/50	774.9	0.5850	1.0599	156.5	7/15	p(219)	(−)YDHAIR
10/14	774.9	0.5825	1.0553	247.9	9/15	p(217)	(−)YHDAIR

Example 2

The amino acid sequence of Peptide Y, a known, standard peptide, was determined using the method of the invention, as applied to Peptide X in Example 1. Peptide Y has the following amino acid sequence: YGGFIRR (SEQ ID NO:265). The molecular mass of Peptide Y was determined to be 868.4719 to an experimental accuracy of 30 ppm from the mass spectrum shown in FIG. 6 . The masses at 1296.6854 and 1570.6774 are from internal standards, added to allow instrument calibration.

The set of amino acids that are possibly part of Peptide Y were then defined for consideration in the analysis. The defined set of amino acids with the molecular mass of each amino acid less the mass of the one water molecule lost during peptide bond formation are the same as those used in Example 1.

As the mass of Peptide Y was measured as 868.4719 to an experimental accuracy of ±30 ppm, each allowed amino acid combination must therefore have a total mass equal to 868.4719±30 ppm. In addition, from the immonium ion region of the PSD trace from FIG. 6 , shown in FIG. 7 , it was determined that Peptide Y must also contain the following amino acids: tyrosine with a characteristic mass at 136.027, phenylalanine with a characteristic mass at 120.071, arginine with a characteristic mass at 175.00, and leucine or isoleucine with a characteristic mass at 85.9225.

Application of the equation in Example 1 demonstrated that only the following combinations of amino acids are allowed for Peptide Y:

1) Tyrosine, phenylalanine, arginine, asparagine, and arginine.

2) Tyrosine, phenylalanine, arginine, arginine, leucine/isoleucine, glycine, and glycine.

3) Tyrosine, phenylalanine, arginine, leucine/isoleucine, alanine, alanine, and glutamine.

4) Tyrosine, phenylalanine, arginine, leucine/isoleucine, glycine, valine, and asparagine

5) Tyrosine, phenylalanine, arginine, leucine/isoleucine, glycine, glycine, glycine, and valine

6) Tyrosine, phenylalanine, arginine, leucine/isoleucine, glycine, alanine, alanine, and alanine

These combinations constitute the allowed set of amino acid combinations for Peptide Y.

In addition, Peptide Y was obtained by a tryptic cleavage, and, thus, from the accepted specificity of trypsin, Peptide Y must also have lysine or arginine as its carboxy terminal amino acid. With this constraint, the allowed library of linear peptides for Peptide Y is constructed from all individual linear permutations of the combinations above. The allowed library includes over 20,000 peptides, and is thus not shown.

As with Example 1, the method of U.S. Pat. No. 5,538,897 was then used to match Peptide Y to this library by tandem mass spectrometry. The experimental tandem mass spectrum of Peptide Y is shown in FIG. 8 , and the top 10 ranking peptides matched to this spectrum are given below (SEQ ID NOS:265-274). Of these ten, the top ranking peptide, YGGFIRR is known to be Peptide Y.

Rank/	(M +					Refer-
Sp	H)	Cn	C * 10 4	Sp	Ions	ence	Peptide
1/3	868.5	1.000	1.894	376.6	11/24	p(415)	(−)YGGFIRR
2/1	868.5	0.967	1.831	440.4	11/24	p(298)	(−)YGGRIFR
3/15	868.5	0.966	1.830	322.8	11/28	p(1975)	(−)YGGFIGVR
4/15	868.5	0.965	1.828	322.8	11/28	p(1735)	(−)YGGFIVGR
5/5	868.5	0.961	1.821	361.7	11/24	p(454)	(−)YGGRFIR
6/2	868.5	0.960	1.819	408.0	11/24	p(1311)	(−)YGVNIFR
7/12	868.5	0.951	1.802	333.7	11/24	p(1527)	(−)YGVNFIR
8/8	868.5	0.942	1.783	356.9	11/28	p(2153)	(−)YGGGVIFR
9/13	868.5	0.937	1.775	331.0	11/24	p(394)	(−)YGGIFRR
10/8	868.5	0.935	1.771	356.9	11/28	p(2147)	(−)YGGVGIFR

Example 3

The amino acid sequence of Peptide Z, a known standard peptide, was determined using the method of the invention, as applied to Peptide X in Example 1 and Peptide Y in Example 2. Peptide Z has the following amino acid sequence: RPPGFSPFR (SEQ ID NO:344). The molecular mass of Peptide Z was determined to be 1060.5660 to an experimental accuracy of 30 ppm from the mass spectrum shown in FIG. 9 . The masses at 1181.6477, 1296.6933 and 1570.6774 are from internal standards, added to allow instrument calibration.

The set of amino acids that are possibly part of Peptide Z were then defined for consideration in the analysis. The defined set of amino acids with the molecular mass of each amino acid less the mass of the one water molecule lost during peptide bond formation are the same as those used in Examples 1 and 2.

As the mass of Peptide Z was measured as 1060.5660 to an experimental accuracy of 30 ppm, each allowed amino acid combination must therefore sum to a mass equal to 1060.5660±30 ppm. In addition, from the immonium ion region of the PSD trace from FIG. 9 , shown in FIG. 10 , it was determined that Peptide Z must also contain the following amino acids: phenylalanine with a characteristic mass at 120.20, arginine with a characteristic mass at 174.94, serine together with proline as deduced from the mass at 167.23, and glycine together with proline as deduced from the mass at 155.66.

Application of the equation in Example 1 was used to determine the allowed combinations of amino acids for Peptide Z, and demonstrates that only the following combinations of amino acids are allowed for Peptide Y (SEQ ID NOS:275-343):

PTIW+FRPSG	GAAAAR+FRPSG	GVVNK+FRPSG	AVVID+FRPSG
VVIW+FRPSG	GPPVF+FRPSG	ASPNK+FRPSG	SPVVD+FRPSG
GQRR+FRPSG	GPVIM+FRPSG	GGAAIK+FRPSG	GVINN+FRPSG
ANRR+FRPSG	APVVM+FRPSG	GGGPTK+FRPSG	AVVNN+FRPSG
GOARR+FRPSG	AAIIE+FRPSG	GGGVVK+FRPSG	GGGVIN+FRPSG
PPFR+FRPSG.	GPTIE+FRPSG	GAAAVK+FRPSG	GAAAIN+FRPSG
PIMR+FRPSG	GVVIE+FRPSG	GGASPK+FRPSG	GGAVVN+FRPSG
VIER+FRPSG	ASPIE+FRPSG	IQQQ+FRPSG	AAAAVN+FRPSG
VNQR+FRPSG	APVTE+FRPSG	GAIQQ+FRPSG	SSPII+FRPSG
CGVQR+FRPSG	AVVVE+FRPSG	AAVQQ+FRPSG	SPVTI+FRPSG
AAAQR+FRPSG	GSPKK+FRPSG	AAINQ+FRPSG	GGGGGVI+FRPSG
IIDR+FRPSG	IQQK+FRPSG	GVVNQ+FRPSG	GGGAAAAI+FRPSG
INM+FRPSG	GAIQK+FRPSG	GGAAIQ+FRPSG	PPTTT+FRPSG
GGINR+FRPSG	AAVQK+FRPSG	GGGVVQ+FRPSG	PVVTT+FRPSG
GAVNR+FRPSG	GSPQK+FRPSG	AAAVQ+FRPSG	GGGGAVV+FRPSG
GGGGIR+FRPSG	AAINY+FRPSG	GVIID+FRPSG	GGAAAAV+FRPSG
GGGAVR+FRPSG	GPTNK+FRPSG	APTID+FRPSG	AAAAAAA+FRPSG

These combinations constitute the allowed set of amino acid combinations for Peptide Z.

In addition, Peptide Z was obtained by a tryptic cleavage, and, from the accepted specificity of trypsin, Peptide Z must have lysine or arginine as its carboxy terminal amino acid. With this constraint, the allowed library of linear peptides for Peptide Z is constructed from all individual linear permutations of the combinations above. The allowed library includes over 2,000,000 peptides, and is thus not shown.

As with Examples 1 and 2, the method of U.S. Pat. No. 5,538,897 was then used to match Peptide Z to this library by tandem mass spectrometry. The experimental tandem mass spectrum of Peptide Z is shown in FIG. 11 , and the top 10 ranking peptides matched to this spectrum provided below (SEQ ID NOS:344-353). Of these ten, the top ranking peptide, RPPGFSPFR (SEQ ID NO:344) is known to be Peptide Z.

Rank/	(M +		C *			Refer-
Sp	H)	Cn	10 4	Sp	Ions	ence     Peptide
1/1	1061.2	1.000	3.310	1163.5	19/24	p(135) (−)RPPGFSPFR
2/2	1061.2	0.871	2.884	1126.6	19/24	p(120) (−)RPPGFPSFR
3/5	1061.2	0.857	2.835	824.7	17/24	p(122) (−)RPPFGPSFR
4/11	1061.2	0.849	2.811	692.8	16/24	p(164) (−)RPPGFFPSR
5/4	1061.2	0.833	2.759	831.2	17/24	p(189) (−)RPPGFFSPR
6/3	1061.2	0.831	2.749	872.9	17/24	p(131) (−)RPPSFGPFR
7/6	1061.2	0.819	2.711	797.1	17/24	p(126) (−)RPFGPPSFR
8/12	1061.2	0.806	2.668	674.0	16/24	p(100) (−)RPPGPSFFR
9/13	1061.2	0.792	2.623	668.4	16/24	p(137) (−)RFPPGSPFR
10/14	1061.2	0.782	2.588	656.5	16/24	p(138) (−)RFGPPSPFR

While it is apparent that the invention disclosed herein is well calculated to fulfill the objectives stated above, it will be appreciated that numerous modifications and embodiments may be devised by those skilled in the art. Therefore, it is intended that the appended claims cover all such modifications and embodiments that fall within the true spirit and scope of the present invention.

353 1 5 PRT Artificial Sequence Peptide X Library 1Tyr Ile His Trp Arg 1 5 2 5 PRT Artificial Sequence Peptide X Library 2Ile Tyr His Trp Arg 1 5 3 5 PRT Artificial Sequence Peptide X Library 3Tyr His Ile Trp Arg 1 5 4 5 PRT Artificial Sequence Peptide X Library 4His Tyr Ile Trp Arg 1 5 5 5 PRT Artificial Sequence Peptide X Library 5Ile His Tyr Trp Arg 1 5 6 5 PRT Artificial Sequence Peptide X Library 6His Ile Tyr Trp Arg 1 5 7 5 PRT Artificial Sequence Peptide X Library 7Tyr Ile Trp His Arg 1 5 8 5 PRT Artificial Sequence Peptide X Library 8Ile Tyr Trp His Arg 1 5 9 5 PRT Artificial Sequence Peptide X Library 9Tyr Trp Ile His Arg 1 5 10 5 PRT Artificial Sequence Peptide X Library 10Trp Tyr Ile His Arg 1 5 11 5 PRT Artificial Sequence Peptide X Library 11Ile Trp Tyr His Arg 1 5 12 5 PRT Artificial Sequence Peptide X Library 12Trp Ile Tyr His Arg 1 5 13 5 PRT Artificial Sequence Peptide X Library 13Tyr His Trp Ile Arg 1 5 14 5 PRT Artificial Sequence Peptide X Library 14His Tyr Trp Ile Arg 1 5 15 5 PRT Artificial Sequence Peptide X Library 15Tyr Trp His Ile Arg 1 5 16 5 PRT Artificial Sequence Peptide X Library 16Trp Tyr His Ile Arg 1 5 17 5 PRT Artificial Sequence Peptide X Library 17His Trp Tyr Ile Arg 1 5 18 5 PRT Artificial Sequence Peptide X Library 18Trp His Tyr Ile Arg 1 5 19 5 PRT Artificial Sequence Peptide X Library 19Ile His Trp Tyr Arg 1 5 20 5 PRT Artificial Sequence Peptide X Library 20His Ile Trp Tyr Arg 1 5 21 5 PRT Artificial Sequence Peptide X Library 21Ile Trp His Tyr Arg 1 5 22 5 PRT Artificial Sequence Peptide X Library 22Trp Ile His Tyr Arg 1 5 23 5 PRT Artificial Sequence Peptide X Library 23His Trp Ile Tyr Arg 1 5 24 5 PRT Artificial Sequence Peptide X Library 24Trp His Ile Tyr Arg 1 5 25 6 PRT Artificial Sequence Peptide X Library 25Tyr Ile His Glu Gly Arg 1 5 26 6 PRT Artificial Sequence Peptide X Library 26Ile Tyr His Glu Gly Arg 1 5 27 6 PRT Artificial Sequence Peptide X Library 27Tyr His Ile Glu Gly Arg 1 5 28 6 PRT Artificial Sequence Peptide X Library 28His Tyr Ile Glu Gly Arg 1 5 29 6 PRT Artificial Sequence Peptide X Library 29Ile His Tyr Glu Gly Arg 1 5 30 6 PRT Artificial Sequence Peptide X Library 30His Ile Tyr Glu Gly Arg 1 5 31 6 PRT Artificial Sequence Peptide X Library 31Tyr Ile Glu His Gly Arg 1 5 32 6 PRT Artificial Sequence Peptide X Library 32Ile Tyr Glu His Gly Arg 1 5 33 6 PRT Artificial Sequence Peptide X Library 33Tyr Glu Ile His Gly Arg 1 5 34 6 PRT Artificial Sequence Peptide X Library 34Glu Tyr Ile His Gly Arg 1 5 35 6 PRT Artificial Sequence Peptide X Library 35Ile Glu Tyr His Gly Arg 1 5 36 6 PRT Artificial Sequence Peptide X Library 36Glu Ile Tyr His Gly Arg 1 5 37 6 PRT Artificial Sequence Peptide X Library 37Tyr His Glu Ile Gly Arg 1 5 38 6 PRT Artificial Sequence Peptide X Library 38His Tyr Glu Ile Gly Arg 1 5 39 6 PRT Artificial Sequence Peptide X Library 39Tyr Glu His Ile Gly Arg 1 5 40 6 PRT Artificial Sequence Peptide X Library 40Glu Tyr His Ile Gly Arg 1 5 41 6 PRT Artificial Sequence Peptide X Library 41His Glu Tyr Ile Gly Arg 1 5 42 6 PRT Artificial Sequence Peptide X Library 42Glu His Tyr Ile Gly Arg 1 5 43 6 PRT Artificial Sequence Peptide X Library 43Ile His Glu Tyr Gly Arg 1 5 44 6 PRT Artificial Sequence Peptide X Library 44His Ile Glu Tyr Gly Arg 1 5 45 6 PRT Artificial Sequence Peptide X Library 45Ile Glu His Tyr Gly Arg 1 5 46 6 PRT Artificial Sequence Peptide X Library 46Glu Ile His Tyr Gly Arg 1 5 47 6 PRT Artificial Sequence Peptide X Library 47His Glu Ile Tyr Gly Arg 1 5 48 6 PRT Artificial Sequence Peptide X Library 48Glu His Ile Tyr Gly Arg 1 5 49 6 PRT Artificial Sequence Peptide X Library 49Tyr Ile His Gly Glu Arg 1 5 50 6 PRT Artificial Sequence Peptide X Library 50Ile Tyr His Gly Glu Arg 1 5 51 6 PRT Artificial Sequence Peptide X Library 51Tyr His Ile Gly Glu Arg 1 5 52 6 PRT Artificial Sequence Peptide X Library 52His Tyr Ile Gly Glu Arg 1 5 53 6 PRT Artificial Sequence Peptide X Library 53Ile His Tyr Gly Glu Arg 1 5 54 6 PRT Artificial Sequence Peptide X Library 54His Ile Tyr Gly Glu Arg 1 5 55 6 PRT Artificial Sequence Peptide X Library 55Tyr Ile Gly His Glu Arg 1 5 56 6 PRT Artificial Sequence Peptide X Library 56Ile Tyr Gly His Glu Arg 1 5 57 6 PRT Artificial Sequence Peptide X Library 57Tyr Gly Ile His Glu Arg 1 5 58 6 PRT Artificial Sequence Peptide X Library 58Gly Tyr Ile His Glu Arg 1 5 59 6 PRT Artificial Sequence Peptide X Library 59Ile Gly Tyr His Glu Arg 1 5 60 6 PRT Artificial Sequence Peptide X Library 60Gly Ile Tyr His Glu Arg 1 5 61 6 PRT Artificial Sequence Peptide X Library 61Tyr His Gly Ile Glu Arg 1 5 62 6 PRT Artificial Sequence Peptide X Library 62His Tyr Gly Ile Glu Arg 1 5 63 6 PRT Artificial Sequence Peptide X Library 63Tyr Gly His Ile Glu Arg 1 5 64 6 PRT Artificial Sequence Peptide X Library 64Gly Tyr His Ile Glu Arg 1 5 65 6 PRT Artificial Sequence Peptide X Library 65His Gly Tyr Ile Glu Arg 1 5 66 6 PRT Artificial Sequence Peptide X Library 66Gly His Tyr Ile Glu Arg 1 5 67 6 PRT Artificial Sequence Peptide X Library 67Ile His Gly Tyr Glu Arg 1 5 68 6 PRT Artificial Sequence Peptide X Library 68His Ile Gly Tyr Glu Arg 1 5 69 6 PRT Artificial Sequence Peptide X Library 69Ile Gly His Tyr Glu Arg 1 5 70 6 PRT Artificial Sequence Peptide X Library 70Gly Ile His Tyr Glu Arg 1 5 71 6 PRT Artificial Sequence Peptide X Library 71His Gly Ile Tyr Glu Arg 1 5 72 6 PRT Artificial Sequence Peptide X Library 72Gly His Ile Tyr Glu Arg 1 5 73 6 PRT Artificial Sequence Peptide X Library 73Tyr Ile Glu Gly His Arg 1 5 74 6 PRT Artificial Sequence Peptide X Library 74Ile Tyr Glu Gly His Arg 1 5 75 6 PRT Artificial Sequence Peptide X Library 75Tyr Glu Ile Gly His Arg 1 5 76 6 PRT Artificial Sequence Peptide X Library 76Glu Tyr Ile Gly His Arg 1 5 77 6 PRT Artificial Sequence Peptide X Library 77Ile Glu Tyr Gly His Arg 1 5 78 6 PRT Artificial Sequence Peptide X Library 78Glu Ile Tyr Gly His Arg 1 5 79 6 PRT Artificial Sequence Peptide X Library 79Tyr Ile Gly Glu His Arg 1 5 80 6 PRT Artificial Sequence Peptide X Library 80Ile Tyr Gly Glu His Arg 1 5 81 6 PRT Artificial Sequence Peptide X Library 81Tyr Gly Ile Glu His Arg 1 5 82 6 PRT Artificial Sequence Peptide X Library 82Gly Tyr Ile Glu His Arg 1 5 83 6 PRT Artificial Sequence Peptide X Library 83Ile Gly Tyr Glu His Arg 1 5 84 6 PRT Artificial Sequence Peptide X Library 84Gly Ile Tyr Glu His Arg 1 5 85 6 PRT Artificial Sequence Peptide X Library 85Tyr Glu Gly Ile His Arg 1 5 86 6 PRT Artificial Sequence Peptide X Library 86Glu Tyr Gly Ile His Arg 1 5 87 6 PRT Artificial Sequence Peptide X Library 87Tyr Gly Glu Ile His Arg 1 5 88 6 PRT Artificial Sequence Peptide X Library 88Gly Tyr Glu Ile His Arg 1 5 89 6 PRT Artificial Sequence Peptide X Library 89Glu Gly Tyr Ile His Arg 1 5 90 6 PRT Artificial Sequence Peptide X Library 90Gly Glu Tyr Ile His Arg 1 5 91 6 PRT Artificial Sequence Peptide X Library 91Ile Glu Gly Tyr His Arg 1 5 92 6 PRT Artificial Sequence Peptide X Library 92Glu Ile Gly Tyr His Arg 1 5 93 6 PRT Artificial Sequence Peptide X Library 93Ile Gly Glu Tyr His Arg 1 5 94 6 PRT Artificial Sequence Peptide X Library 94Gly Ile Glu Tyr His Arg 1 5 95 6 PRT Artificial Sequence Peptide X Library 95Glu Gly Ile Tyr His Arg 1 5 96 6 PRT Artificial Sequence Peptide X Library 96Gly Glu Ile Tyr His Arg 1 5 97 6 PRT Artificial Sequence Peptide X Library 97Tyr His Glu Gly Ile Arg 1 5 98 6 PRT Artificial Sequence Peptide X Library 98His Tyr Glu Gly Ile Arg 1 5 99 6 PRT Artificial Sequence Peptide X Library 99Tyr Glu His Gly Ile Arg 1 5 100 6 PRT Artificial Sequence Peptide X Library 100Glu Tyr His Gly Ile Arg 1 5 101 6 PRT Artificial Sequence Peptide X Library 101His Glu Tyr Gly Ile Arg 1 5 102 6 PRT Artificial Sequence Peptide X Library 102Glu His Tyr Gly Ile Arg 1 5 103 6 PRT Artificial Sequence Peptide X Library 103Tyr His Gly Glu Ile Arg 1 5 104 6 PRT Artificial Sequence Peptide X Library 104His Tyr Gly Glu Ile Arg 1 5 105 6 PRT Artificial Sequence Peptide X Library 105Tyr Gly His Glu Ile Arg 1 5 106 6 PRT Artificial Sequence Peptide X Library 106Gly Tyr His Glu Ile Arg 1 5 107 6 PRT Artificial Sequence Peptide X Library 107His Gly Tyr Glu Ile Arg 1 5 108 6 PRT Artificial Sequence Peptide X Library 108Gly His Tyr Glu Ile Arg 1 5 109 6 PRT Artificial Sequence Peptide X Library 109Tyr Glu Gly His Ile Arg 1 5 110 6 PRT Artificial Sequence Peptide X Library 110Glu Tyr Gly His Ile Arg 1 5 111 6 PRT Artificial Sequence Peptide X Library 111Tyr Gly Glu His Ile Arg 1 5 112 6 PRT Artificial Sequence Peptide X Library 112Gly Tyr Glu His Ile Arg 1 5 113 6 PRT Artificial Sequence Peptide X Library 113Glu Gly Tyr His Ile Arg 1 5 114 6 PRT Artificial Sequence Peptide X Library 114Gly Glu Tyr His Ile Arg 1 5 115 6 PRT Artificial Sequence Peptide X Library 115His Glu Gly Tyr Ile Arg 1 5 116 6 PRT Artificial Sequence Peptide X Library 116Glu His Gly Tyr Ile Arg 1 5 117 6 PRT Artificial Sequence Peptide X Library 117His Gly Glu Tyr Ile Arg 1 5 118 6 PRT Artificial Sequence Peptide X Library 118Gly His Glu Tyr Ile Arg 1 5 119 6 PRT Artificial Sequence Peptide X Library 119Glu Gly His Tyr Ile Arg 1 5 120 6 PRT Artificial Sequence Peptide X Library 120Gly Glu His Tyr Ile Arg 1 5 121 6 PRT Artificial Sequence Peptide X Library 121Ile His Glu Gly Tyr Arg 1 5 122 6 PRT Artificial Sequence Peptide X Library 122His Ile Glu Gly Tyr Arg 1 5 123 6 PRT Artificial Sequence Peptide X Library 123Ile Glu His Gly Tyr Arg 1 5 124 6 PRT Artificial Sequence Peptide X Library 124Glu Ile His Gly Tyr Arg 1 5 125 6 PRT Artificial Sequence Peptide X Library 125His Glu Ile Gly Tyr Arg 1 5 126 6 PRT Artificial Sequence Peptide X Library 126Glu His Ile Gly Tyr Arg 1 5 127 6 PRT Artificial Sequence Peptide X Library 127Ile His Gly Glu Tyr Arg 1 5 128 6 PRT Artificial Sequence Peptide X Library 128His Ile Gly Glu Tyr Arg 1 5 129 6 PRT Artificial Sequence Peptide X Library 129Ile Gly His Glu Tyr Arg 1 5 130 6 PRT Artificial Sequence Peptide X Library 130Gly Ile His Glu Tyr Arg 1 5 131 6 PRT Artificial Sequence Peptide X Library 131His Gly Ile Glu Tyr Arg 1 5 132 6 PRT Artificial Sequence Peptide X Library 132Gly His Ile Glu Tyr Arg 1 5 133 6 PRT Artificial Sequence Peptide X Library 133Ile Glu Gly His Tyr Arg 1 5 134 6 PRT Artificial Sequence Peptide X Library 134Glu Ile Gly His Tyr Arg 1 5 135 6 PRT Artificial Sequence Peptide X Library 135Ile Gly Glu His Tyr Arg 1 5 136 6 PRT Artificial Sequence Peptide X Library 136Gly Ile Glu His Tyr Arg 1 5 137 6 PRT Artificial Sequence Peptide X Library 137Glu Gly Ile His Tyr Arg 1 5 138 6 PRT Artificial Sequence Peptide X Library 138Gly Glu Ile His Tyr Arg 1 5 139 6 PRT Artificial Sequence Peptide X Library 139His Glu Gly Ile Tyr Arg 1 5 140 6 PRT Artificial Sequence Peptide X Library 140Glu His Gly Ile Tyr Arg 1 5 141 6 PRT Artificial Sequence Peptide X Library 141His Gly Glu Ile Tyr Arg 1 5 142 6 PRT Artificial Sequence Peptide X Library 142Gly His Glu Ile Tyr Arg 1 5 143 6 PRT Artificial Sequence Peptide X Library 143Glu Gly His Ile Tyr Arg 1 5 144 6 PRT Artificial Sequence Peptide X Library 144Gly Glu His Ile Tyr Arg 1 5 145 6 PRT Artificial Sequence Peptide X Library 145Tyr Ile His Asp Ala Arg 1 5 146 6 PRT Artificial Sequence Peptide X Library 146Ile Tyr His Asp Ala Arg 1 5 147 6 PRT Artificial Sequence Peptide X Library 147Tyr His Ile Asp Ala Arg 1 5 148 6 PRT Artificial Sequence Peptide X Library 148His Tyr Ile Asp Ala Arg 1 5 149 6 PRT Artificial Sequence Peptide X Library 149Ile His Tyr Asp Ala Arg 1 5 150 6 PRT Artificial Sequence Peptide X Library 150His Ile Tyr Asp Ala Arg 1 5 151 6 PRT Artificial Sequence Peptide X Library 151Tyr Ile Asp His Ala Arg 1 5 152 6 PRT Artificial Sequence Peptide X Library 152Ile Tyr Asp His Ala Arg 1 5 153 6 PRT Artificial Sequence Peptide X Library 153Tyr Asp Ile His Ala Arg 1 5 154 6 PRT Artificial Sequence Peptide X Library 154Asp Tyr Ile His Ala Arg 1 5 155 6 PRT Artificial Sequence Peptide X Library 155Ile Asp Tyr His Ala Arg 1 5 156 6 PRT Artificial Sequence Peptide X Library 156Asp Ile Tyr His Ala Arg 1 5 157 6 PRT Artificial Sequence Peptide X Library 157Tyr His Asp Ile Ala Arg 1 5 158 6 PRT Artificial Sequence Peptide X Library 158His Tyr Asp Ile Ala Arg 1 5 159 6 PRT Artificial Sequence Peptide X Library 159Tyr Asp His Ile Ala Arg 1 5 160 6 PRT Artificial Sequence Peptide X Library 160Asp Tyr His Ile Ala Arg 1 5 161 6 PRT Artificial Sequence Peptide X Library 161His Asp Tyr Ile Ala Arg 1 5 162 6 PRT Artificial Sequence Peptide X Library 162Asp His Tyr Ile Ala Arg 1 5 163 6 PRT Artificial Sequence Peptide X Library 163Ile His Asp Tyr Ala Arg 1 5 164 6 PRT Artificial Sequence Peptide X Library 164His Ile Asp Tyr Ala Arg 1 5 165 6 PRT Artificial Sequence Peptide X Library 165Ile Asp His Tyr Ala Arg 1 5 166 6 PRT Artificial Sequence Peptide X Library 166Asp Ile His Tyr Ala Arg 1 5 167 6 PRT Artificial Sequence Peptide X Library 167His Asp Ile Tyr Ala Arg 1 5 168 6 PRT Artificial Sequence Peptide X Library 168Asp His Ile Tyr Ala Arg 1 5 169 6 PRT Artificial Sequence Peptide X Library 169Tyr Ile His Ala Asp Arg 1 5 170 6 PRT Artificial Sequence Peptide X Library 170Ile Tyr His Ala Asp Arg 1 5 171 6 PRT Artificial Sequence Peptide X Library 171Tyr His Ile Ala Asp Arg 1 5 172 6 PRT Artificial Sequence Peptide X Library 172His Tyr Ile Ala Asp Arg 1 5 173 6 PRT Artificial Sequence Peptide X Library 173Ile His Tyr Ala Asp Arg 1 5 174 6 PRT Artificial Sequence Peptide X Library 174His Ile Tyr Ala Asp Arg 1 5 175 6 PRT Artificial Sequence Peptide X Library 175Tyr Ile Ala His Asp Arg 1 5 176 6 PRT Artificial Sequence Peptide X Library 176Ile Tyr Ala His Asp Arg 1 5 177 6 PRT Artificial Sequence Peptide X Library 177Tyr Ala Ile His Asp Arg 1 5 178 6 PRT Artificial Sequence Peptide X Library 178Ala Tyr Ile His Asp Arg 1 5 179 6 PRT Artificial Sequence Peptide X Library 179Ile Ala Tyr His Asp Arg 1 5 180 6 PRT Artificial Sequence Peptide X Library 180Ala Ile Tyr His Asp Arg 1 5 181 6 PRT Artificial Sequence Peptide X Library 181Tyr His Ala Ile Asp Arg 1 5 182 6 PRT Artificial Sequence Peptide X Library 182His Tyr Ala Ile Asp Arg 1 5 183 6 PRT Artificial Sequence Peptide X Library 183Tyr Ala His Ile Asp Arg 1 5 184 6 PRT Artificial Sequence Peptide X Library 184Ala Tyr His Ile Asp Arg 1 5 185 6 PRT Artificial Sequence Peptide X Library 185His Ala Tyr Ile Asp Arg 1 5 186 6 PRT Artificial Sequence Peptide X Library 186Ala His Tyr Ile Asp Arg 1 5 187 6 PRT Artificial Sequence Peptide X Library 187Ile His Ala Tyr Asp Arg 1 5 188 6 PRT Artificial Sequence Peptide X Library 188His Ile Ala Tyr Asp Arg 1 5 189 6 PRT Artificial Sequence Peptide X Library 189Ile Ala His Tyr Asp Arg 1 5 190 6 PRT Artificial Sequence Peptide X Library 190Ala Ile His Tyr Asp Arg 1 5 191 6 PRT Artificial Sequence Peptide X Library 191His Ala Ile Tyr Asp Arg 1 5 192 6 PRT Artificial Sequence Peptide X Library 192Ala His Ile Tyr Asp Arg 1 5 193 6 PRT Artificial Sequence Peptide X Library 193Tyr Ile Asp Ala His Arg 1 5 194 6 PRT Artificial Sequence Peptide X Library 194Ile Tyr Asp Ala His Arg 1 5 195 6 PRT Artificial Sequence Peptide X Library 195Tyr Asp Ile Ala His Arg 1 5 196 6 PRT Artificial Sequence Peptide X Library 196Asp Tyr Ile Ala His Arg 1 5 197 6 PRT Artificial Sequence Peptide X Library 197Ile Asp Tyr Ala His Arg 1 5 198 6 PRT Artificial Sequence Peptide X Library 198Asp Ile Tyr Ala His Arg 1 5 199 6 PRT Artificial Sequence Peptide X Library 199Tyr Ile Ala Asp His Arg 1 5 200 6 PRT Artificial Sequence Peptide X Library 200Ile Tyr Ala Asp His Arg 1 5 201 6 PRT Artificial Sequence Peptide X Library 201Tyr Ala Ile Asp His Arg 1 5 202 6 PRT Artificial Sequence Peptide X Library 202Ala Tyr Ile Asp His Arg 1 5 203 6 PRT Artificial Sequence Peptide X Library 203Ile Ala Tyr Asp His Arg 1 5 204 6 PRT Artificial Sequence Peptide X Library 204Ala Ile Tyr Asp His Arg 1 5 205 6 PRT Artificial Sequence Peptide X Library 205Tyr Asp Ala Ile His Arg 1 5 206 6 PRT Artificial Sequence Peptide X Library 206Asp Tyr Ala Ile His Arg 1 5 207 6 PRT Artificial Sequence Peptide X Library 207Tyr Ala Asp Ile His Arg 1 5 208 6 PRT Artificial Sequence Peptide X Library 208Ala Tyr Asp Ile His Arg 1 5 209 6 PRT Artificial Sequence Peptide X Library 209Asp Ala Tyr Ile His Arg 1 5 210 6 PRT Artificial Sequence Peptide X Library 210Ala Asp Tyr Ile His Arg 1 5 211 6 PRT Artificial Sequence Peptide X Library 211Ile Asp Ala Tyr His Arg 1 5 212 6 PRT Artificial Sequence Peptide X Library 212Asp Ile Ala Tyr His Arg 1 5 213 6 PRT Artificial Sequence Peptide X Library 213Ile Ala Asp Tyr His Arg 1 5 214 6 PRT Artificial Sequence Peptide X Library 214Ala Ile Asp Tyr His Arg 1 5 215 6 PRT Artificial Sequence Peptide X Library 215Asp Ala Ile Tyr His Arg 1 5 216 6 PRT Artificial Sequence Peptide X Library 216Ala Asp Ile Tyr His Arg 1 5 217 6 PRT Artificial Sequence Peptide X Library 217Tyr His Asp Ala Ile Arg 1 5 218 6 PRT Artificial Sequence Peptide X Library 218His Tyr Asp Ala Ile Arg 1 5 219 6 PRT Artificial Sequence Peptide X Library 219Tyr Asp His Ala Ile Arg 1 5 220 6 PRT Artificial Sequence Peptide X Library 220Asp Tyr His Ala Ile Arg 1 5 221 6 PRT Artificial Sequence Peptide X Library 221His Asp Tyr Ala Ile Arg 1 5 222 6 PRT Artificial Sequence Peptide X Library 222Asp His Tyr Ala Ile Arg 1 5 223 6 PRT Artificial Sequence Peptide X Library 223Tyr His Ala Asp Ile Arg 1 5 224 6 PRT Artificial Sequence Peptide X Library 224His Tyr Ala Asp Ile Arg 1 5 225 6 PRT Artificial Sequence Peptide X Library 225Tyr Ala His Asp Ile Arg 1 5 226 6 PRT Artificial Sequence Peptide X Library 226Ala Tyr His Asp Ile Arg 1 5 227 6 PRT Artificial Sequence Peptide X Library 227His Ala Tyr Asp Ile Arg 1 5 228 6 PRT Artificial Sequence Peptide X Library 228Ala His Tyr Asp Ile Arg 1 5 229 6 PRT Artificial Sequence Peptide X Library 229Tyr Asp Ala His Ile Arg 1 5 230 6 PRT Artificial Sequence Peptide X Library 230Asp Tyr Ala His Ile Arg 1 5 231 6 PRT Artificial Sequence Peptide X Library 231Tyr Ala Asp His Ile Arg 1 5 232 6 PRT Artificial Sequence Peptide X Library 232Ala Tyr Asp His Ile Arg 1 5 233 6 PRT Artificial Sequence Peptide X Library 233Asp Ala Tyr His Ile Arg 1 5 234 6 PRT Artificial Sequence Peptide X Library 234Ala Asp Tyr His Ile Arg 1 5 235 6 PRT Artificial Sequence Peptide X Library 235His Asp Ala Tyr Ile Arg 1 5 236 6 PRT Artificial Sequence Peptide X Library 236Asp His Ala Tyr Ile Arg 1 5 237 6 PRT Artificial Sequence Peptide X Library 237His Ala Asp Tyr Ile Arg 1 5 238 6 PRT Artificial Sequence Peptide X Library 238Ala His Asp Tyr Ile Arg 1 5 239 6 PRT Artificial Sequence Peptide X Library 239Asp Ala His Tyr Ile Arg 1 5 240 6 PRT Artificial Sequence Peptide X Library 240Ala Asp His Tyr Ile Arg 1 5 241 6 PRT Artificial Sequence Peptide X Library 241Ile His Asp Ala Tyr Arg 1 5 242 6 PRT Artificial Sequence Peptide X Library 242His Ile Asp Ala Tyr Arg 1 5 243 6 PRT Artificial Sequence Peptide X Library 243Ile Asp His Ala Tyr Arg 1 5 244 6 PRT Artificial Sequence Peptide X Library 244Asp Ile His Ala Tyr Arg 1 5 245 6 PRT Artificial Sequence Peptide X Library 245His Asp Ile Ala Tyr Arg 1 5 246 6 PRT Artificial Sequence Peptide X Library 246Asp His Ile Ala Tyr Arg 1 5 247 6 PRT Artificial Sequence Peptide X Library 247Ile His Ala Asp Tyr Arg 1 5 248 6 PRT Artificial Sequence Peptide X Library 248His Ile Ala Asp Tyr Arg 1 5 249 6 PRT Artificial Sequence Peptide X Library 249Ile Ala His Asp Tyr Arg 1 5 250 6 PRT Artificial Sequence Peptide X Library 250Ala Ile His Asp Tyr Arg 1 5 251 6 PRT Artificial Sequence Peptide X Library 251His Ala Ile Asp Tyr Arg 1 5 252 6 PRT Artificial Sequence Peptide X Library 252Ala His Ile Asp Tyr Arg 1 5 253 6 PRT Artificial Sequence Peptide X Library 253Ile Asp Ala His Tyr Arg 1 5 254 6 PRT Artificial Sequence Peptide X Library 254Asp Ile Ala His Tyr Arg 1 5 255 6 PRT Artificial Sequence Peptide X Library 255Ile Ala Asp His Tyr Arg 1 5 256 6 PRT Artificial Sequence Peptide X Library 256Ala Ile Asp His Tyr Arg 1 5 257 6 PRT Artificial Sequence Peptide X Library 257Asp Ala Ile His Tyr Arg 1 5 258 6 PRT Artificial Sequence Peptide X Library 258Ala Asp Ile His Tyr Arg 1 5 259 6 PRT Artificial Sequence Peptide X Library 259His Asp Ala Ile Tyr Arg 1 5 260 6 PRT Artificial Sequence Peptide X Library 260His Ala Asp Ile Tyr Arg 1 5 261 6 PRT Artificial Sequence Peptide X Library 261His Ala Asp Ile Tyr Arg 1 5 262 6 PRT Artificial Sequence Peptide X Library 262Ala His Asp Ile Tyr Arg 1 5 263 6 PRT Artificial Sequence Peptide X Library 263Asp Ala His Ile Tyr Arg 1 5 264 6 PRT Artificial Sequence Peptide X Library 264Ala Asp His Ile Tyr Arg 1 5 265 7 PRT Artificial Sequence Peptide Y Library 265Tyr Gly Gly Phe Ile Arg Arg 1 5 266 7 PRT Artificial Sequence Peptide Y Library 266Tyr Gly Gly Arg Ile Phe Arg 1 5 267 8 PRT Artificial Sequence Peptide Y Library 267Tyr Gly Gly Phe Ile Gly Val Arg 1 5 268 8 PRT Artificial Sequence Peptide Y Library 268Tyr Gly Gly Phe Ile Val Gly Arg 1 5 269 7 PRT Artificial Sequence Peptide Y Library 269Tyr Gly Gly Arg Phe Ile Arg 1 5 270 7 PRT Artificial Sequence Peptide Y Library 270Tyr Gly Val Asn Ile Phe Arg 1 5 271 7 PRT Artificial Sequence Peptide Y Library 271Tyr Gly Val Asn Phe Ile Arg 1 5 272 6 PRT Artificial Sequence Peptide Y Library 272Tyr Gly Gly Gly Val Ile 1 5 273 7 PRT Artificial Sequence Peptide Y Library 273Tyr Gly Gly Ile Phe Arg Arg 1 5 274 8 PRT Artificial Sequence Peptide Y Library 274Tyr Gly Gly Val Gly Ile Phe Arg 1 5 275 4 PRT Artificial Sequence Peptide Z Library 275Pro Thr Ile Trp 1 276 5 PRT Artificial Sequence Peptide Z Library 276Phe Arg Pro Ser Gly 1 5 277 4 PRT Artificial Sequence Peptide Z Library 277Val Val Ile Trp 1 278 4 PRT Artificial Sequence Peptide Z Library 278Gly Gln Arg Arg 1 279 4 PRT Artificial Sequence Peptide Z Library 279Ala Asn Arg Arg 1 280 4 PRT Artificial Sequence Peptide Z Library 280Gly Ala Arg Arg 1 281 4 PRT Artificial Sequence Peptide Z Library 281Pro Pro Phe Arg 1 282 4 PRT Artificial Sequence Peptide Z Library 282Pro Ile Met Arg 1 283 4 PRT Artificial Sequence Peptide Z Library 283Val Ile Glu Arg 1 284 4 PRT Artificial Sequence Peptide Z Library 284Val Asn Gln Arg 1 285 5 PRT Artificial Sequence Peptide Z Library 285Cys Gly Val Gln Arg 1 5 286 5 PRT Artificial Sequence Peptide Z Library 286Ala Ala Ala Gln Arg 1 5 287 4 PRT Artificial Sequence Peptide Z Library 287Ile Ile Asp Arg 1 288 3 PRT Artificial Sequence Peptide Z Library 288Ile Asn Met 1 289 5 PRT Artificial Sequence Peptide Z Library 289Gly Gly Ile Asn Arg 1 5 290 5 PRT Artificial Sequence Peptide Z Library 290Gly Ala Val Asn Arg 1 5 291 6 PRT Artificial Sequence Peptide Z Library 291Gly Gly Gly Gly Ile Arg 1 5 292 6 PRT Artificial Sequence Peptide Z Library 292Gly Gly Gly Ala Val Arg 1 5 293 6 PRT Artificial Sequence Peptide Z Library 293Gly Ala Ala Ala Ala Arg 1 5 294 5 PRT Artificial Sequence Peptide Z Library 294Gly Pro Pro Val Phe 1 5 295 5 PRT Artificial Sequence Peptide Z Library 295Gly Pro Val Ile Met 1 5 296 5 PRT Artificial Sequence Peptide Z Library 296Ala Pro Val Val Met 1 5 297 5 PRT Artificial Sequence Peptide Z Library 297Ala Ala Ile Ile Glu 1 5 298 5 PRT Artificial Sequence Peptide Z Library 298Gly Pro Thr Ile Glu 1 5 299 5 PRT Artificial Sequence Peptide Z Library 299Gly Val Val Ile Glu 1 5 300 5 PRT Artificial Sequence Peptide Z Library 300Ala Ser Pro Ile Glu 1 5 301 5 PRT Artificial Sequence Peptide Z Library 301Ala Pro Val Thr Glu 1 5 302 5 PRT Artificial Sequence Peptide Z Library 302Ala Val Val Val Glu 1 5 303 5 PRT Artificial Sequence Peptide Z Library 303Gly Ser Pro Lys Lys 1 5 304 4 PRT Artificial Sequence Peptide Z Library 304Ile Gln Gln Lys 1 305 5 PRT Artificial Sequence Peptide Z Library 305Gly Ala Ile Gln Lys 1 5 306 5 PRT Artificial Sequence Peptide Z Library 306Ala Ala Val Gln Lys 1 5 307 5 PRT Artificial Sequence Peptide Z Library 307Gly Ser Pro Gln Lys 1 5 308 5 PRT Artificial Sequence Peptide Z Library 308Ala Ala Ile Asn Tyr 1 5 309 5 PRT Artificial Sequence Peptide Z Library 309Gly Pro Thr Asn Lys 1 5 310 5 PRT Artificial Sequence Peptide Z Library 310Gly Val Val Asn Lys 1 5 311 5 PRT Artificial Sequence Peptide Z Library 311Ala Ser Pro Asn Lys 1 5 312 6 PRT Artificial Sequence Peptide Z Library 312Gly Gly Ala Ala Ile Lys 1 5 313 6 PRT Artificial Sequence Peptide Z Library 313Gly Gly Gly Pro Thr Lys 1 5 314 6 PRT Artificial Sequence Peptide Z Library 314Gly Gly Gly Val Val Lys 1 5 315 6 PRT Artificial Sequence Peptide Z Library 315Gly Ala Ala Ala Val Lys 1 5 316 6 PRT Artificial Sequence Peptide Z Library 316Gly Gly Ala Ser Pro Lys 1 5 317 4 PRT Artificial Sequence Peptide Z Library 317Ile Gln Gln Gln 1 318 5 PRT Artificial Sequence Peptide Z Library 318Gly Ala Ile Gln Gln 1 5 319 5 PRT Artificial Sequence Peptide Z Library 319Ala Ala Val Gln Gln 1 5 320 5 PRT Artificial Sequence Peptide Z Library 320Ala Ala Ile Asn Gln 1 5 321 5 PRT Artificial Sequence Peptide Z Library 321Gly Val Val Asn Gln 1 5 322 6 PRT Artificial Sequence Peptide Z Library 322Gly Gly Ala Ala Ile Gln 1 5 323 6 PRT Artificial Sequence Peptide Z Library 323Gly Gly Gly Val Val Gln 1 5 324 5 PRT Artificial Sequence Peptide Z Library 324Ala Ala Ala Val Gln 1 5 325 5 PRT Artificial Sequence Peptide Z Library 325Gly Val Ile Ile Asp 1 5 326 5 PRT Artificial Sequence Peptide Z Library 326Ala Pro Thr Ile Asp 1 5 327 5 PRT Artificial Sequence Peptide Z Library 327Ala Val Val Ile Asp 1 5 328 5 PRT Artificial Sequence Peptide Z Library 328Ser Pro Val Val Asp 1 5 329 5 PRT Artificial Sequence Peptide Z Library 329Gly Val Ile Asn Asn 1 5 330 5 PRT Artificial Sequence Peptide Z Library 330Ala Val Val Asn Asn 1 5 331 6 PRT Artificial Sequence Peptide Z Library 331Gly Gly Gly Val Ile Asn 1 5 332 6 PRT Artificial Sequence Peptide Z Library 332Gly Ala Ala Ala Ile Asn 1 5 333 6 PRT Artificial Sequence Peptide Z Library 333Gly Gly Ala Val Val Asn 1 5 334 6 PRT Artificial Sequence Peptide Z Library 334Ala Ala Ala Ala Val Asn 1 5 335 5 PRT Artificial Sequence Peptide Z Library 335Ser Ser Pro Ile Ile 1 5 336 5 PRT Artificial Sequence Peptide Z Library 336Ser Pro Val Thr Ile 1 5 337 7 PRT Artificial Sequence Peptide Z Library 337Gly Gly Gly Gly Gly Val Ile 1 5 338 8 PRT Artificial Sequence Peptide Z Library 338Gly Gly Gly Ala Ala Ala Ala Ile 1 5 339 5 PRT Artificial Sequence Peptide Z Library 339Pro Pro Thr Thr Thr 1 5 340 5 PRT Artificial Sequence Peptide Z Library 340Pro Val Val Thr Thr 1 5 341 7 PRT Artificial Sequence Peptide Z Library 341Gly Gly Gly Gly Ala Val Val 1 5 342 7 PRT Artificial Sequence Peptide Z Library 342Gly Gly Ala Ala Ala Ala Val 1 5 343 7 PRT Artificial Sequence Peptide Z Library 343Ala Ala Ala Ala Ala Ala Ala 1 5 344 9 PRT Artificial Sequence Top Ranked Peptide Library 344Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5 345 9 PRT Artificial Sequence Top Ranked Peptide Library 345Arg Pro Pro Gly Phe Pro Ser Phe Arg 1 5 346 9 PRT Artificial Sequence Top Ranked Peptide Library 346Arg Pro Pro Phe Gly Pro Ser Phe Arg 1 5 347 9 PRT Artificial Sequence Top Ranked Peptide Library 347Arg Pro Pro Gly Phe Phe Pro Ser Arg 1 5 348 9 PRT Artificial Sequence Top Ranked Peptide Library 348Arg Pro Pro Gly Phe Phe Ser Pro Arg 1 5 349 9 PRT Artificial Sequence Top Ranked Peptide Library 349Arg Pro Pro Ser Phe Gly Pro Phe Arg 1 5 350 9 PRT Artificial Sequence Top Ranked Peptide Library 350Arg Pro Phe Gly Pro Pro Ser Phe Arg 1 5 351 9 PRT Artificial Sequence Top Ranked Peptide Library 351Arg Pro Pro Gly Pro Ser Phe Phe Arg 1 5 352 9 PRT Artificial Sequence Top Ranked Peptide Library 352Arg Phe Pro Pro Gly Ser Pro Phe Arg 1 5 353 9 PRT Artificial Sequence Top Ranked Peptide Library 353Arg Phe Gly Pro Pro Ser Pro Phe Arg 1 5

Claims

1. A method for determining the amino-acid sequence of a peptide of interest having a molecular mass, the method comprising:(a) designating a set of allowed amino acids; (b) determining the molecular mass of the peptide of interest; (c) collecting an experimental fragmentation mass spectrum for the peptide of interest using a mass spectrometer; (d) defining a set of allowed combinations comprising all combinations of allowed amino acids having a predicted total molecular mass consistent with the molecular mass of the peptide of interest; (e) defining a set of allowed peptides comprising all linear permutations of the allowed combinations; (f) calculating a theoretical fragmentation mass spectrum for allowed peptides in the set of allowed peptides, comprising calculating fragment-ion masses thereof and assigning an intensity value to one or more fragment ions of the theoretical fragmentation mass spectrum; and (g) comparing the experimental fragmentation mass spectrum of the peptide of interest to one or more theoretical fragmentation mass spectra calculated for the allowed peptides to determine the amino-acid sequence of the peptide of interest.

2. The method of claim 1, wherein the mass spectrometer is a time-of-flight mass spectrometer.

3. The method of claim 1, wherein the designated set of allowed amino acids comprises tryptophan, arginine, histidine, glutamic acid, glutamine, aspartic acid, leucine, threonine, proline, alanine, tyrosine, phenylalanine, methionine, lysine, asparagine, isoleucine, cysteine, valine, serine, and glycine.

4. The method of claim 1, wherein the molecular mass of the peptide of interest is determined with an accuracy of about 30 parts per million and the predicted total molecular mass of each allowed combination is within a range of plus or minus about 30 parts per million of the molecular mass of the peptide of interest.

5. The method of claim 1, wherein the peptide of interest has been treated to remove one or more post-translational modifications.

6. The method of claim 1, further comprising calculating an indication of closeness-of-fit between the experimental fragmentation mass spectrum of the peptide of interest and the theoretical fragmentation mass spectra.

7. The method of claim 6, wherein calculating the indication of closeness-of-fit comprises selecting peak values in the theoretical fragmentation mass spectra having an assigned intensity greater than a predetermined threshold value.

8. The method of claim 1, further comprising normalizing the experimental fragmentation mass spectrum.

9. The method of claim 1, wherein the designated set of allowed amino acids comprises carbamido cysteine.

10. The method of clam 1, wherein the peptide of interest has a molecular mass of greater than about 1,400 Daltons.

11. A method for determining the amino-acid sequence of a peptide of interest having a molecular mass, the method comprising:(a) designating a set of allowed amino acids; (b) determining the molecular mass of the peptide of interest; (c) collecting an experimental fragmentation mass spectrum for the peptide of interest using a mass spectrometer; (d) identifying one or more amino acids present in the peptide of interest; (e) defining a set of allowed combinations comprising all combinations of allowed amino acids having a predicted total molecular mass consistent with the molecular mass of the peptide of interest, wherein every allowed combination includes the one or more identified amino acids; (f) defining a set of allowed peptides comprising all linear permutations of the allowed combinations; (g) calculating a theoretical fragmentation mass spectrum for allowed peptides in the set of allowed peptides, comprising calculating fragment-ion masses thereof and assigning an intensity value to one or more fragment ions of the theoretical fragmentation mass spectrum; and (h) comparing the experimental fragmentation mass spectrum of the peptide of interest to one or more theoretical fragmentation mass spectra calculated for the allowed peptides to determine the amino-acid sequence of the peptide of interest.

12. The method of claim 11, wherein the peptide of interest has a molecular mass of greater than about 1,400 Daltons.

13. The method of claim 11, further comprising analyzing an immonium region of the experimental fragmentation mass spectrum to identify one or more amino acids.

14. The method of claim 11, wherein a plurality of identified amino acids form a peptide fragment.

15. The method of claim 11, wherein the designated set of allowed amino acids comprises tryptophan, arginine, histidine, glutamic acid, glutamine, aspartic acid, leucine, threonine, proline, alanine, tyrosine, phenylalanine, methionine, lysine, asparagine, isoleucine, cysteine, valine, serine, and glycine.

16. The method of claim 11, wherein the designated set of allowed amino acids comprises carbamido cysteine.

17. The method of claim 11, wherein the peptide of interest has been treated to remove one or more post-translational modifications.