Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
1999-02-10
2001-09-11
Anderson, Bruce C. (Department: 2878)
Radiant energy
Ionic separation or analysis
Methods
C250S287000
Reexamination Certificate
active
06288389
ABSTRACT:
FIELD OF INVENTION
The invention relates to a method of fast real time evaluation of mass spectra for analytical methods where in thousands of spectra per day the only result to be established is whether previously known mass signals are present or not present.
PRIOR ART
In some areas of analysis there is currently widespread talk of the term “High Sample Through-put” (HST), which is defined as a daily sample throughput of 50,000 to 100,000 samples. Partially by so-called “massive-parallel” processing and partially by very fast sequential measurement and preparation methods the samples are pretreated and measured analytically. For sequential measurements with corresponding data evaluation there are only 1½ seconds per sample available in the case of 50,000 samples a day and only about ¾ of a second for 100,000 samples a day, whereby a slight time buffer has to be included for changing sample batches. Mass spectrometry has so far been regarded as a relatively slow method, not only concerning the evaluation of the spectra, which can certainly take many minutes to a number of hours, but also concerning the measurements. However, the argument of slowness does not necessarily apply. Time-of-flight mass spectrometry, for example, with ionization by matrix-assisted laser desorption (MALDI) can definitely be regarded as one of the candidates for such a high sample throughput technology. Particularly the application of MALDI time-of-flight spectrometry to molecular weight determination of oligonucleotides, but also peptides from enzymatic protein digestive matter, makes such a high sample throughput technology not only desirable but also possible. Another field is the analysis of active products in combinatorial chemistry, for which MALDI methods can also be used.
In the meantime methods have become known for massive-parallel synthesis, sample preparation, sample cleaning, matrix addition, and pipetting onto large sample supports for these MALDI methods. Also there are promising approaches toward the accurate and dense preparation of the samples on the sample supports, and for automated, highly sensitive laser desorption without any visual control with very fast and accurate positioning of the samples in the ion source. The problem is therefore particularly reduced to the data evaluation process, which also has to be conducted in the short time period which is available for analysis if no insuperable data pile-up is to occur.
The raw data of a spectrum consist of individual ion current measurements which have been acquired and digitized at a fixed rate and stored in that sequence. The time values of the measurements are not stored as well—they correspond to the addresses of the measured ion current values in the computer memory. Usually the measurements of several individual spectra are already added together for the raw data in order to improve the signal-to-noise ratio. Sometimes there are also checks between the additions to establish whether the newly recorded individual spectrum meets certain quality requirements before it is added to the sum of the individual spectra recorded so far.
A time-of-flight raw mass spectrum obtained by adding individual raw spectra together with a scna over about 100 microseconds consists of about 200 kilobytes of data at a measuring rate of 1 gigahertz, but with the transient recorders already available nowadays, which have a scanning rate of 4 gigahertz, it would consist of about 800 kilobytes of data. With current transient recorders the reading of data alone requires the available time; future transient recorders (which have already been announced) with very fast data transfer buses may be of assistance though. Consequently the problem can be restricted further: only the peak search and conversion of flight times to masses currently still take many seconds per spectrum. However, as described above, only these ¾ second are available for reading the spectra, assessment, addition, evaluation, and storage of the results.
According to current technology not only one spectrum is scanned in those ¾ second but, as described above, several spectra are measured and added together to improve the signal-to-noise ratio. Since the individual spectra are not always reproducible, each individual spectrum is read, investigated for usability by special methods, and then, upon release, added to the sum of the other spectra. So far it cannot be assumed that each spectrum will automatically succeed and produce sufficient mass resolution. However, promising techniques are being developed which minimize frequent production of outlier spectra or even eliminate them completely.
Consequently, in the brief period of less than one second not only does evaluation have to take place, the spectra must also be scanned and added together. For scanning MALDI time-of-flight mass spectra it is known that frequently well over a hundred individual spectra have to be added together before signals are obtained which can be properly evaluated. The scanning rates are limited to about 20 spectra per second though because otherwise the samples become charged, leading to displacements of the ion signals and therefore spectra which cannot be added together.
Therefore one must endeavor to make do by adding about
10
individual spectra. This in turn makes special complicating demands on the recognition of ion current signals, which under these circumstances are often difficult to distinguish against the background noise.
On the other hand, the analysis methods which serve as target methods for high sample throughput (HST) are usually characterized by the fact that they are limited to few responses of qualitative nature per sample spectrum. For instance, mutation analyses of DNA samples are characterized by the fact that only one or two signals are present in the spectrum, and they can appear at a maximum of four or six precisely known molecular masses. All the other ion current signals in the spectrum are irrelevant: they originate either from the matrix substance which has to be added to the sample, from fragment ions, from dimers or oligomers, or from undesirable additives to the actual analyte substance. In the case of biallelic mutations, signals can in principle only occur at two to four known points. In the analysis of microsatellites a correctly measured signal can be found at one out of a maximum of approximately 30 precisely known points. The analysis of products by combinatorial chemistry can produce signals at one location out of a total in the order of 1,000.
OBJECTIVE OF THE INVENTION
It is the objective of the invention to find a method for the evaluation of mass spectra, particularly MALDI time-of-flight mass spectra, which, on the one hand, can be performed in the very short period of time available and, on the other, also guarantees good detection of the signals even under poor signal-to-noise conditions
BRIEF DESCRIPTION OF THE INVENTION
It is the basic idea of the invention that the inundation of raw data of the mass spectrum, stored in a computer memory, is examined only at known memory addresses (corresponding to flight times in our example) for the occurrence of expectable signals. The raw data are not examined for mass peaks continuously and converted to a mass spectrum via a calibration curve, instead the masses of the expectable ion signals are converted to memory addresses by the inverted calibration curve and the stored measurement data are investigated in a stationary manner at the corresponding addresses as to whether a signal is present or not.
In the following the invention is particularly described by the example of the MALDI time-of-flight mass spectra, without limiting the invention to this type of mass spectra For a serial high sample throughput analytical method, there is not much time available, therefore not very many time-of-flight spectra can be added together, as described above. In addition, MALDI time-of-flight spectra frequently show high background noise resulting from the matrix ions which covers more or less the entire
Anderson Bruce C.
Bruker Daltonik GmbH
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