Radiant energy – Calibration or standardization methods
Reexamination Certificate
2001-05-30
2003-08-19
Berman, Jack (Department: 2881)
Radiant energy
Calibration or standardization methods
Reexamination Certificate
active
06608302
ABSTRACT:
FIELD OF THE INVENTION
The present invention is a method for improving the calibration of a Fourier transform ion cyclotron resonance mass spectrometer wherein the frequency spectrum of a sample has been measured and the frequency (f) and intensity (I) of at least three species having known mass to charge (m/z) ratios and one specie having an unknown (m/z) ratio have been identified. More specifically, the method uses known (m/z) ratios, frequencies, and intensities of at least three species to calculate coefficients, A, B, and C, wherein the mass to charge ratio of at least one of the three species (m/z)
i
is equal to
A
f
i
+
B
f
i
2
+
C
·
G
(
I
i
)
f
i
Q
wherein f
i
is the detected frequency of the specie, G(I
i
) is a predetermined function of the intensity of the specie, and Q is a predetermined exponent. Using the calculated values A, B, and C, the mass to charge ratio of the unknown specie (m/z)
ii
is calculated as the sum of
A
f
ii
+
B
f
ii
2
+
C
·
G
(
I
ii
)
f
ii
Q
wherein f
ii
is the measured frequency of the unknown specie, and (I
ii
) is the measured intensity of the unknown specie.
BACKGROUND OF THE INVENTION
For human understanding of physical, biological, and chemical systems to progress, a need for ever greater accuracy in measuring species becomes a limiting factor for accurate insight into the operation of these systems. For example, with the increased availability of genomic databases, protein identification is now substantially based on searching an appropriate database with physico-chemical data obtained for that protein. Very often, mass spectrometric data from tandem mass spectrometry (MS/MS) experiments using peptides from protein digests are employed. One of the aspects of mass spectrometry, which is often viewed as the key to successful protein identification, is mass measurement accuracy (MMA). Increased mass accuracy allows the number of potential masses in a database to be reduced, and sufficiently high MMA may make a peptide unique within the context of a specific proteome.
Fourier transform ion cyclotron resonance (FTICR) mass spectrometry currently provides the best achievable mass accuracy. However, the mass accuracy in an FTICR experiment typically depends on the number of ions used for the measurement. When online separations are used, the analyte ion production rates vary widely, and the ion population in the trap cannot be easily or precisely controlled. Although mass accuracy in the sub-ppm level has been reported with internal calibration, external calibration methods currently known in the art typically don't provide accuracies better than several ppm, particularly when the ion population for the measurement differs significantly from the ion population used for the calibration. In FTICR, the highest MMA have been obtained with small ion populations, often with the use of summation (or signal averaging) of many spectra, and of internal calibrants. However, if one desires a large dynamic range, large trapped ion populations are desired, which irrevocably causes relatively large space charge induced frequency shifts, and poorer MMA.
The widely varying ion populations that result from online separation constitute the greatest challenge. The difficulties for large ion populations in FTICR arise due to Coulomb mediated interactions between the different ions present in the cell (and their interactions with their image charge on the detection electrodes), which cause variations in measured frequencies. It has recently been demonstrated in Bruce, J. E.; Anderson, G. A.; Brands, M. D.; Pasa-Tolic, L.; and Smith, R. D.
J. Am Soc Mass Spectrom
2000, 11, 416-421 the entire contents of which are incorporated herein by this reference, that the frequency shifts induced by coulombic interactions can be compensated for by correcting the detected frequencies, so as to align the deconvoluted spectrum of multiple charge states of the same peptide or protein. This approach provides most of the advantages associated with internal calibrant without its disadvantages. This procedure has allowed a significant improvement in mass accuracy for peptides in LC/FTMS experiments, but the mass accuracy realized still plateaus at the few ppm level due to the large variations in space charge effects.
All calibration procedures for ICR have, up to now, incorporated the space charge effect as a global effect resulting only from the number of charges in the trap. However, some frequency perturbations are known to depend on the frequency spacing between ions, e.g. the “peak-coalescence” phenomenon. It is clear that the contribution of such smaller effects is obscured by the global space charge effect, and until now, little experimental evidence of “local” frequency perturbations has been reported by Huang, J. Y.; P. W. Tiedemann, Land, D. P.; McIver, R. T; Hemminger, J. C. Int. J. Mass Spectrom. Ion Proc. 1994, 134(1), 11-21, the en ire contents of which are incorporated herein by this reference. Indeed, some authors have suggested that such an effect doesn't exist Easterling, M. L.; Mize, T. H.; Amster, I. J.,
Anal. Chem.
1999, 71, 624-632.
In FTICR, the measured quantity is the effective (cyclotron) frequency of the ions, f. This frequency is then converted to an m/z value using a calibration function. The most widespread used calibration function is (1):
m
z
=
A
f
+
B
f
2
(
1
)
This calibration law (1) was originally derived by Gross and coworkers as reported in Ledford, E. B.; Rempel, D. L.; Gross, M. L.
Anal. Chem.
1984, 56, 2744-2748, the entire contents of which are incorporated herein by this reference, using results as reported in Jeffries, J. B.; Barlow, S. E.; Dunn, G. H.
Int. J. Mass Spectrom. Ion Processes
1983, 54, 169-187 and Francl, T. J.; Sherman, M. G.; Hunter, R. L.; Locke, M. J.; Bowers, W. D.; McIver, R. T. Int.
J. Mass Spectrom, Ion Processes
1983, 54, 189-199 the entire contents of which are also incorporated herein by this reference. According to these references, the derivation of the second term, B/f
2
, accounts for both the DC trapping field and the space charge influence. The space charge is assumed to be generated by all ion species present in the ICR cell during collection of the time domain signal. The two calibration coefficients A and B thus are theorized to account for factors important for the FTICR mass measurement, i.e. magnetic field strength, and radial components of the trapping DC electrostatic field and the space charge field. Although an additional third-order frequency term can be added to the calibration function (1), there are no quantitative reports on its importance for the improvement of calibration quality
This calibration technique assumes that the space charge is generated by all ion species present in the ICR cell during collection of the time domain signal. While this “global” space charge correction has been shown to improve accuracy of the mass calibration under conditions typical for bio-molecular studies, when the ion population in the ICR cell may vary in a broad range, it still suffers from drawbacks that hinder its accuracy. For example, the concept of a “global” space charge correction assumes that only the total trapped ion charge is significant for the mass calibration and fails to account for the possibility that the coherent motion of ions having the same m/z is influenced by other m/z ions differently than by the ions themselves. Such a situation may occur, for example, when the ion cloud motion can be, to a good approximation, described in terms of its center-of-mass motion. In this case the coulombic interactions of the same m/z ions, constituting the ion cloud, will be balanced and will not produce a net effect on the center-of-mass motion of the ion cloud. Under these conditions, accurate mass measurements must account for the coulombic interactions of the same m/z ions, constituting the ion cloud, since they will be balanced and will not produce a net effect on the center-of-mass motion of the ion cloud. Thus, there remains a need for improved me
Masselon Christophe D.
Smith Richard D.
Tolmachev Aleksey
Berman Jack
May Stephen R.
McKinley, Jr. Douglas E.
Smith II Johnnie L
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