Method of determining peptide sequences by mass spectrometry

Details Method of determining peptide sequences by mass spectrometry Method of determining peptide sequences by mass spectrometry

2000-04-06

2002-12-03

Lee, John R. (Department: 2881)

Radiant energy

Ionic separation or analysis

C250S282000

Reexamination Certificate

active

06489608

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
A Method of Determining Peptide Sequences by Mass Spectrometry
This invention relates to methods of determining the sequence of amino acids that constitute peptides, polypeptides or proteins by mass spectrometry and especially by tandem mass spectrometry or MS/MS. In particular it relates to methods whereby the sequence can be determined from the mass spectral data alone and which do not require the use of existing libraries of protein sequence information. Methods according to the invention require no information concerning the nature of the peptide other than a library of the amino acid residues that may occur in proteins weighted according to natural abundance.
2. Discussion of the Prior Art
Although several well-established chemical methods for the sequencing of peptides, polypeptides and proteins are known (for example, the Edman degradation), mass spectrometric methods are becoming increasingly important in view of their speed and ease of use. Mass spectrometric methods have been developed to the point at which they are capable of sequencing peptides in a mixture without any prior chemical purification or separation, typically using electrospray ionization and tandem mass spectrometry (MS/MS). For example, see Yates III (J. Mass Spectrom, 1998 vol. 33 pp. 1-19), Papayannopoulos (Mass Spectrom. Rev. 1995, vol. 14 pp. 49-73), and Yates III, McCormack, and Eng (Anal. Chem. 1996 vol. 68 (17) pp. 534A-540A). Thus, in a typical MS/MS sequencing experiment, molecular ions of a particular peptide are selected by the first mass analyzer and fragmented by collisions with neutral gas molecules in a collision cell. The second mass analyzer is then used to record the fragment ion spectrum that generally contains enough information to allow at least a partial, and often the complete, sequence to be determined.
Unfortunately, however, the interpretation of the fragment spectra is not straightforward. Manual interpretation (see, for example, Hunt, Yates III, et al, Proc. Nat. Acad. Sci. USA, 1986, vol. 83 pp 6233-6237 and Papayannopoulos, ibid) requires considerable experience and is time consuming. Consequently, many workers have developed algorithms and computer programs to automate the process, at least in part. The nature of the problem, however, is such that none of those so far developed are able to provide in reasonable time complete sequence information without either requiring some prior knowledge of the chemical structure of the peptide or merely identifying likely candidate sequences in existing protein structure databases. The reason for this will be understood from the following discussion of the nature of the fragment spectra produced.
Typically, the fragment spectrum of a peptide comprises peaks belonging to about half a dozen different ion series each of which correspond to different modes of fragmentation of the peptide parent ion. Each typically (but not invariably) comprises peaks representing the loss of successive amino acid residues from the original peptide ion. Because all but two of the 20 amino acids from which most naturally occurring proteins are comprised have different masses, it is therefore possible to establish the sequence of amino acids from the difference in mass of peaks in any given series which correspond to the successive loss of an amino acid residue from the original peptide. However, difficulties arise in identifying to which series an ion belongs and from a variety of ambiguities that can arrive in assigning the peaks, particularly when certain peaks are either missing or unrecognized. Moreover, other peaks are typically present in a spectrum due to various more complicated fragmentation or rearrangement routes, so that direct assignment of ions is fraught with difficulty. Further, electrospray ionization tends to produce multiply charged ions that appear at correspondingly resealed masses, which further complicates the interpretation of the spectra. Isotopic clusters also lead to proliferation of peaks in the observed spectra. Thus, the direct transformation of a mass spectrum to a sequence is only possible in trivially small peptides.
The reverse route, transforming trial sequences to predicted spectra for comparison with the observed spectrum, should be easier, but has not been fully developed. The number of possible sequences for any peptide (20
n
, where n is the number of amino acids comprised in the peptide) is very large, so the difficulty of finding the correct sequence for, say, a peptide of a mere 10 amino acids (20
10
=10
13
possible sequences) will be appreciated. The number of potential sequences increases very rapidly both with the size of the peptide and with the number (at least 20) of the residues being considered.
Details of the first computer programs for predicting probable amino acid sequences from mass spectral data appeared in 1984 (Sakurai, Matsuo, Matsuda, Katakuse, Biomed. Mass Spectrom, 1984, vol. 11 (8) pp 397-399). This program (PAAS3) searched through all the amino acid sequences whose molecular weights coincided with that of the peptide being examined and identified the most probable sequences with the experimentally observed spectra. Hamm, Wilson and Harvan (CABIOS, 1986 vol. 2 (2) pp 115-118) also developed a similar program.
However, as pointed out by Ishikawa and Niwa (Biomed. and Environ. Mass Spectrom. 1986, vol. 13 pp 373-380), this approach is limited to peptides not exceeding 800 daltons in view of the computer time required to carry out the search. Parekh et al in UK patent application 2,325,465 (published November 1998) have resurrected this idea and give an example of the sequencing of a peptide of 1000 daltons which required 2×10
6
possible sequences to be searched, but do not specify the computer time required. Nevertheless, despite the increase in the processing speed of computers between 1984 and 1999, a simple search of all possible sequences for a peptide of molecular weights greater than 1200 daltons is still impractical in a reasonable time using the personal computer typically supplied for data processing with most commercial mass spectrometers.
This problem has long been recognized and many attempts have been made to render the problem more tractable. For example, the MS/MS spectrum may be correlated with amino acid sequences derived from a protein database rather than every possible sequence. Such methods are taught in PCT patent application 95/25281, by Taylor and Johnson (Rapid Commun. in Mass Spectrom. 1997 vol. 11 pp 1067-1075, by Eng. McCormack, Yates in J. Am. Mass Spectrom. 1994 vol. 5 pp 976-989, by Figeys, Lock et al. (Rapid Commun. in Mass Spectrom. 1998 vol. 12 pp 1435-1444), and by Mortz, O'Connor et al (Proc. Nat. Acid Sci. USA 1996 vol. 93 pp 8264-8267). Alternatively, MS/MS experiments can be carried out on both the original peptide and a derivative of it, and the results from both experiments combined to establish at least a partial sequence without reference to a database. (See, for example, the isotopic labeling method taught by Shevchenko, Chernushevich et al in Rapid Commun. in Mass Spectrom, 1997 vol. 11 pp 1015-24, or the esterification method taught by Yates III, Griffin and Hood in Techniques in Protein Chem. II, ch 46 (1991) pp 477-485), and the H
2
/D
2
exchange method taught by Septov, Issakova et al in Rapid Commun. in Mass Spectrom. 1993 vol. 7 pp 58-62. Johnson and Walsh (Protein Science, 1992 vol. 1 pp 1083-1091) teach a similar method, combining Edman degradation data and MS/MS data.
Of the prior programs which attempt to predict sequence information using only MS/MS data and without reference to existing databases, a variety of methods have been suggested to facilitate the prediction of sequence information. Siegel and Bauman (Biomed. Environ. Mass Spectrom. 1998 vol. 15 pp 333-343) describe an algorithm which builds up the sequence information stepwise from the mass difference between neighbouring ions in ion series recognized in the spectrum, but good results were obtained only wi

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