Mass spectral peak identification

Details Mass spectral peak identification Mass spectral peak identification

2002-10-30

2004-06-01

Barlow, John (Department: 2863)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Chemical analysis

Reexamination Certificate

active

06745133

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to the identification of peaks of a mass spectrum obtained by mass spectrometry, and more specifically to a method for determining a measure of the mono-isotopic mass of a molecule such as a polymer or a bio-molecule.
TECHNICAL BACKGROUND OF THE INVENTION
The mass of a molecule, such as a bio-molecule or a polymer, can be determined using mass spectrometry. With this method a sample for analysis is ionized and analysed in a mass spectrometer to determine a mass spectroscopic data set, which usually is presented as a mass spectrum. The mass spectrum exhibits intensity peaks that are associated with the mass, or more specifically with the mass-to-charge ratio, of the molecule.
The technology associated with mass spectroscopy is well known, and is thoroughly described in numerous publications such as “Mass Spectrometry Principles and Applications”, E. De Hoffmann, J. Charette, V. Stroobart; John Wiley & Sons Ltd, Chichester and “Mass Spectrometry of Biological Materials”, B. S. Larsen, C. N. McEvans, Marcel Dekker Inc., New York.
It should be pointed out, that depending on the equipment used the mass estimation obtained could relate either to a molecule or to an ion. In the case that the mass spectrometer ionizes the molecule by adding hydrogen ions, the mass obtained should be reduced with the weight of the charge carrying hydrogen ions. However, for simplicity of the description, and since this circumstance is well known within the art, only the term “molecule” will be used below.
Today, the most common way to determine the position, i.e. the mass-to-charge ratio, of an individual peak is probably the “centroiding method”. With this method, after having isolated a specific peak of the mass spectrum a start point SP at the positive slope of the peak (“the low mass end”) and an end point EP at the negative slope (“the high mass end”) are determined. Using a geometrical analogy, the top of the peak is defined as that mass to charge value between SP and EP that represents a point of balance of the peak area above a line between SP and EP.
When used for mixed low and high masses, the centroiding method generates peak positions that are both average masses and monoisotopic masses. Thus, for identification of the compound that caused a specific peak, one must also make further analysis to determine if the value is an average value or a monoisotopic value.
However, this method has a number of drawbacks. For example, when analysing heavy molecules, the peaks of separate isotopes will merge due to the limited resolution of the instrument. Therefore, the centroiding method will yield an average molecule mass.
When analysing a molecule of comparatively low mass, the resolution of the instrument is often sufficient to allow the centroiding method to be used for determining a monoisotopic mass. If such a molecule is used to calibrate the instrument together with the centroiding method, a systematic error will be introduced when analysing heavier molecules, for which the peaks are not resolved. This occurs since the use of the centroiding method for the heavier molecules yields an average mass, as described above, which differs from the monoisotopic mass, i.e. the average mass is always higher than the monoisotopic mass.
In addition, at low concentrations the signal-to-noise ratio becomes low. Therefore, it will be difficult to identify a proper start point SP and end point EP respectively, on which the centroiding method is based.
Furthermore, the centroiding method has a limited sensitivity to the shape of the intensity curve between the low and the high end of the measuring interval.
However, in many circumstances it is of interest to determine the monoisotopic molecular mass of the sample molecule, i.e. the mass of a molecule consisting only of the lowest mass isotopes. For reasons given above, the known methods to analyse a mass spectrum, herein represented by the centroiding method, are not well adapted to determine the monoisotopic molecular mass based on a mass spectrum having badly resolved isotopic peaks. Similarly, the centroiding method is not well adapted to a case wherein the peaks of a heavy molecule are well resolved in them selves, but the intensities of the isotopes of relatively low mass are near the noise level of the signal.
In GB-2,333,893 A (Bruker) there is disclosed a method based on mass spectrometry suitable for accurate determination of unknown ions. This method uses a curve fitting method and a mathematial optimization method to find a best fit between a model spectrum and a measured spectrum. However, it does not adress the problem of unknown m/Z values, for a single family of isotopic peaks.
Therefore, there is a need for an improved method for determining the monoisotopic molecular mass, including the molecular mass-to-charge ratio, of a molecule analysed by mass spectrometry.
SUMMARY OF THE INVENTION
It is an object of the present invention to meet this need. This object is achieved with a method according to claim
1
of the appended claims.
With the method of the invention it is possible to determine the monoisotopic molecular mass-to-charge ratio of a sample molecule with a considerable reliability.
Having determined the monoisotopic molecular mass-to-charge ratio, the monoisotopic mass is obtained by simply multiplying by the number of the associated charge state.
In a specific embodiment of the method of the invention, the method is extended to include determination of the charge state in a case where the charge state is unknown, thereby also allowing monoisotopic mass determination in such a case.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention are given by way of illustration only. Various changes and modifications within the inventive idea and scope of the invention will become apparent to those skilled in the art from this detailed description.


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A. Henderson, et al. “Computer peak identification in SIMS and comparison with XPS chemical state identification. EMAG-MICRO 89”, Proceedings of the Institute of Physics Electron Microscopy and Analysis Group and Royal Microscopy Society Conference, London, UK, Sep. 13-15, 1989, pp. 335-338, vol. 1.
C. L. Do Lago, et al. “New method of isotope pattern analysis”, Computers Chem. vol. 15, No. 2, pp. 149-155, 1991, UK.

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