Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
1999-01-28
2001-02-13
Anderson, Bruce C. (Department: 2878)
Radiant energy
Ionic separation or analysis
Methods
C250S281000
Reexamination Certificate
active
06188064
ABSTRACT:
The invention relates to accurate determinations of the position coordinate, i.e. the location, frequency, or time coordinate of an ion peak in a mass spectrum, as the basis for an accurate mass determination of the ions.
The invention consists of fitting a suitable function, e.g. a superposition of bell-shaped curves, to all peaks of a peak group simultaneously instead of using one isolated mass peak only, and applying a suitable abundance distribution for all the peaks of the measured group, e.g. of an isotopic pattern. The true mass distances between the individual peaks of the peak group and the ratio of their widths are known with high accuracy. The distances between the peaks of an isotopic pattern can, for instance, be derived in a good approximation from mean compositions of the substances of a chemical class. During curve fitting by a mathematical optimization process, in the simplest case only the position coordinate and the width of the bell-shaped curves are varied. If the pattern used is an isotopic pattern, a precise position determination of the monoisotopic ions of the isotope group is obtained automatically, even if the monoisotopic peak is not visible at all. For organic substances, in the simplest case only the pattern of the carbon isotopes in that chemical class are used for pattern fitting.
STATE OF THE ART
For accurate mass determination of ions of an unknown substance using a mass spectrometer, the mass spectrometer always first has to be calibrated with a known calibration substance, preferably at several points on the mass spectrum. By this calibration procedure a function called a “calibration curve” is obtained between the position of an ion peak in the mass spectrum and the mass of the ions of that ion peak. Then a spectrum of an unknown substance can be measured and the mass of an unknown ion can be calculated using this the calibration curve. For more accurate measurements one adds to the unknown substance one or two known reference substances and corrects the masses of the unknown substance using the mass differences, which one has found for the reference substances between calculated and true masses (method with “internal reference”).
The basis of all these calibration and measurement methods is always a calculation of a location, frequency, or time coordinate for an individual ion mass peak in the mass spectrum, which consists in total of a large number of individual digital measurement values. Location coordinates are obtained in spectra of static mass spectrometers with spatial resolution using photographic plates or diode arrays, frequency values in Fourier transform mass spectrometers, time values in time-scan mass spectrometers and in time-of-flight mass spectrometers. In doing so one must derive the accurate location, frequency, or time value from a measured (local or temporal) profile of the measured values across a mass peak. In the simplest case a centroid formation of the individual measured values is used. In slightly more elaborate but slightly more accurate methods a theoretically derived function is fitted into the measurement profile of a mass peak, from which the optimal position of the location, frequency or time value are derived.
In the following, the location, frequency, or time coordinates are only referred to as the “position coordinates”, or simply to the “position” of a mass peak in the mass spectrum.
This determination of the position of the signal profile of an ion in this spectrum, however, constitutes the main source of inaccuracies of mass determination. Since this determination of the position is used both in calibration and in the measurement of the unknown substance, the error increases in proportion.
An attempt to increase the accuracy of mass determination is therefore frequently undertaken by scanning a very large number of mass peaks along the spectrum during calibration and evaluating them, and then through their position coordinates, adapting a smooth curve, levelling out some inaccuracies. Since the same method for the measurement of the unknown mass cannot be used because the substance shows only a single peak, the gain in accuracy is limited. For more complex ions (heavy organic ions, for instance) the peak of an ion is always accompanied by several peaks of the same elemental but different isotopic composition. This is called here an “isotope group” of ion peaks.
In this case one can determine the mass of all these ions individually and therefrom calculate an improved mean for one peak of the pattern. If the atomic composition of the ion is known, one can very accurately calculate the correct mass distances between the ion peaks necessary for this averaging procedure. However, the accuracies of mass determination achieved so far are not yet satisfactory.
Improved accuracy of mass calculation is particularly important for the reliable identification of proteins. Here a protein usually is digested by an enzyme (trypsin for example), whereby the protein is always cut adjacent to amino acids specific to the enzyme. In this way digestion products are obtained which are about 10 to 20 amino acids long on a statistical average, but the length of which is naturally considerably dispersed and ranges from 1 to about 40 amino acids. Thus they cover a mass range of 100 to 5,000 atomic mass units. For this mass range an improved accuracy of mass determination is urgently being sought to be able to use the results of improved mass determination to identify the protein by referring to protein data banks more accurately. In this mass range good mass spectrometers can still resolve the ion peaks of an isotope group which therefore are separated fairly well (such a resolution is normally referred to as “unit mass resolution”).
Nevertheless, an improvement is also being sought for the subsequent range of approximately 5,000 to 10,000 atomic mass units. Good time-of-flight mass spectrometers, for instance, can also provide unit mass resolution in that range.
For ions in this mass range of 5,000 to 10,000 atomic mass units there is a further difficulty for mass calibration: the mass peak of the so-called “monoisotopic” ions which is comprised of atoms of each of the most frequent isotopes, can no longer be easily identified and usually it is no longer visible in this spectrum. For instance, in the case of bovine insulin (the mass of the monoisotopic peak is 5731.616 atomic mass units) one frequently does not see the protonated, monoisotopic molecule peak of the compound
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, at least not unless the spectrum is excellent and has a very good signal-to-noise ratio. Of the many hundred peaks of the abundance distribution of the isotopic pattern, which up to
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covers a total of 872 mass units, one sees only about 10 peaks from the distribution maximum.
Therefore, even with known substances it is not at all easy to actually allocate the individual peaks to the correct isotope compounds and therefore to the true masses. Calibration errors can easily occur in this way. Determination of the monoisotopic mass for unknown ions is even more difficult.
OBJECTIVE OF THE INVENTION
It is the objective of the invention to find a method for a precise determination of the position of ion mass signals which is superior to the methods of centroid determination or curve fitting of individual mass signals and which can be used as a basis for precise calibration and measurement processes. It is a secondary objective of the invention to achieve automatic recognition of the position of the monoisotopic peak for heavy ions.
BRIEF DESCRIPTION OF THE INVENTION
It is the basic idea of the invention to simultaneously fit a whole family of bell-shaped curves of known mass distances, known abundances and known width ratios into a measured signal pattern of several ion peaks, instead to fit just one single bell-shaped function curve into a measured signal profile of a single ion peak, as done previously. For the method according to this invention it is necessary to know the true
Anderson Bruce C.
Bruker Daltonik GmbH
Wells Nikita
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