Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-04-06
2001-12-04
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S968000
Reexamination Certificate
active
06326163
ABSTRACT:
The present invention relates to a method according to the generic part of claim
1
.
Mutations on the gene level often cause inappropriate amino acids to be incorporated in proteins which are encoded in the corresponding gene segment which has mutated. This may result in pathologic phenomena, e.g., sickle-cell anaemia. Examinations of the presence of mutations which are responsible for pathologic conditions are usually performed on the DNA level. Such examinations are cumbersome, and their evaluation takes very long as a rule.
Thus, an object of the invention has been to provide a method by which the presence of mutations can be established quickly and reliably.
According to the invention, this object is achieved by a method with the features as defined in claim
1
.
The method according to the invention serves to recognize mutations in organisms by comparing a deviation, Caused by a mutation, of the amino acid composition of a protein expressed in the region of the mutation (protein to be examined) with a corresponding protein which is expressed by a wild type lacking the mutation. The method according to the invention is characterized in that
a sample is taken from the organism at a site where the protein to be examined is expressed, can be detected and/or plays a physiological role;
either the protein is concentrated or purified by methods of protein analysis, followed by a determination of its molecular weight, or
a determination of the molecular weight of the protein to be examined is performed without a pretreatment of the sample.
The invention advantageously relates to a quick method for a qualitative and quantitative medical-diagnostic analysis on the protein level of the substitution of single amino acids with pathogenic or non-pathogenic effects on the organism. The medical-diagnostic analysis is preferably performed by a combination of enzymatic or chemical cleavage of the isolated peptide, chromatographical separation of the fragments and analysis by mass spectrometry, both direct LC/MS and indirect MALDI-MS, and analysis, e.g., by capillary electrophoresis. By comparing protein samples from healthy subjects with those of ill subjects, the method described is suitable for establishing new, as yet unknown mutations and quantifying the expression and incorporation of wild type to mutant.
The obtaining of samples from an organism, especially by biopsy, can be considered a basis of the method according to the invention. As the sites where the samples are taken, sites are selected in which the protein to be examined is expressed, detectable or plays a physiological role, e.g., in muscle fiber bundles or tissue pieces from organs, especially hearts. The samples are advantageously processed in such a way that the substitution of a single amino acid can be unambiguously detected by mass-spectrometric, chromatographical and/or electrophoretical methods, and the expression and incorporation ratio of wild type to mutant can be quantitatively determined. Thus, it becomes possible by a direct protein analysis as well to diagnose the causes of a genetically caused disease at an early stage and to identify as yet unknown mutations by a comparative analysis of samples from healthy subjects and samples from ill subjects.
By using the highly sensitive “matrix-assisted laser-induced desorption and ionization time-of-flight mass spectrometry” (MALDI-MS) in combination with liquid chromatography, especially using columns with an inner diameter of ≦1 mm (microbore and capillary columns), the detection can be effected even with minute sample quantities.
As compared to the as yet performed analyses of the DNA coding for the proteins, the method is characterized in that the detection of the mutation and the determination of the expression and incorporation ratio is effected in a significantly shorter period of time. An analysis can be performed within one week, in contrast to the usual times required for DNA analysis, ranging from several weeks to half a year.
It is an advantage that a specific cleavage of the protein recovered from biopsy samples for diagnostic purposes is performed with selective enzymes, endoproteinases, or by chemical reagents, and the fragments obtained by such cleavage are separated by chromatographical methods and characterized in terms of their molecular weights either directly (LC/MS) or indirectly (MALDI-MS). The distribution in the separation and the molecular weight, determined by mass spectrometry, is unambiguous evidence for each fragment of the protein.
With smaller proteins, i.e., those having a molecular weight of up to 100,000 Dalton, the amino acid substitutions can be directly determined with the method described, without a preliminary cleavage into fragments. The accuracy of the measurement enables the precise determination of deviations in molecular weight of <5 Dalton and thus the detection of both wild type and mutant in the presence of each other. The precise localization of the amino acid substitution can be effected by the above described cleavage.
Thus, it is possible to characterize all proteins involved in a physiologically functional structure, e.g., muscle, in terms of genetically caused pathogenic and non-pathogenic amino acid substitutions.
Since the amino acid sequences of a lot of proteins are already known due to the sequencing of the human genome and all human proteins will be elucidated in a few years, a protein can be analyzed for mutations by means of the method herein described by selecting the appropriate cleavage which can be achieved by one skilled in the art by per se known methods.
It can be of great importance that this method may also be adapted for clinical-diagnostic use in the recognition of the causes of diseases and, through an optimization of the separation and detection conditions, also for the systematic screening of patient samples for particular, defined mutations.
Using appropriate equipment, the method described may also be automated to a large extent. For the procedure as described, the duration of an analysis is in the range of a few days whereas DNA analysis takes several weeks to months. At the same time, all proteins involved in a physiologically functional structure can be analyzed by this method.
Unlike the conventional methods for the detection of point mutations by analyzing the genomic DNA or cDNA, this method allows the quantification of the expression and incorporation of the mutant in the physiologically functional structure, especially if, due to the presence of a diploid set of chromosomes in the nucleus, both forms of the respective protein are expressed and it is not known in what ratio the two forms are present in the functional form.
The invention further allows evidence of the identity of the wild type and of the mutant to be furnished by chemical sequence analysis or by amino acid analysis.
A preferred embodiment of the method according to the invention comprises the cleavage of the protein to be examined with suitable enzymes or chemical reagents, and a combination of chromatographical methods, especially high-pressure liquid chromatography with capillary columns (MB-HPLC), and mass spectrometry.
The invention will be further illustrated by using the heavy chain of the &bgr;-isoform of myosin as an example. Point mutations in the heavy chain of the ⊖-isoform of myosin, e.g., substitution of the amino acid methionine for the amino acid valine in position 606, may result in hypertrophic cardiomyopathy, a genetically caused thickening of certain heart walls which may lead to sudden death. According to the invention, the detection of the mutation is possible by a combination of enzymatic cleavage and LC/MS.
REFERENCES:
patent: 5208458 (1993-05-01), Busch et al.
patent: 5429923 (1995-07-01), Seidman et al.
patent: WO 95/25281 (1995-03-01), None
patent: WO 95/33856 (1995-06-01), None
Eckardstein et al.,The Journal of Biological Chemistry, “Structural Analysis of Human Apolipoprotein A-I Variants”, vol. 265, No. 15, pp. 8610-8617, 1990.
Falick et al,Rapid Communications in Mass Spe
Brenner Bernhard
Forssmann Wolf-Georg
Nier Volker
Raida Manfred
Jacobson & Holman PLLC
Leary Louise N.
Pharis Biotec GmbH
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