WO2001002848A1

WO2001002848A1 - Method for the multi-dimensional analysis of a proteome

Info

Publication number: WO2001002848A1
Application number: PCT/DE2000/002154
Authority: WO
Inventors: Thomas Moore; Anton Horn; Stefan Kreusch
Original assignee: Thomas Moore; Anton Horn; Stefan Kreusch
Priority date: 1999-07-05
Filing date: 2000-07-04
Publication date: 2001-01-11
Also published as: AU6555500A; GB2367894A; DE10081888A5; DE10081888B4; GB0202280D0; DE19932270A1; GB2367894B

Abstract

The invention relates to a method for the multi-dimensional analysis of a proteome. The method is used in the biochemical, biotechnological and medical fields and in the pharmaceutical industry for diagnostic purposes and for developing biologically active substances. The aim of the invention is to improve, facilitate and for certain proteins first of all to enable the quantification and identification of the proteins of a proteome. According to the invention, the proteins of a proteome are subjected to a large number n of different separation processes under standardised conditions in such a way, that each respective liquid fraction m1, obtained in a separation stage, delivers m2 liquid fractions in a separation stage which immediately follows. After n separation stages, m1* m2* ....mn = M liquid fractions have been produced which are identified qualitatively or quantitatively by known identification methods using o different analysis methods and which are quantitatively determined also by known quantification methods in such a way, that once the analysis data has been unified in a database, an n-dimensional image of the proteome is obtained which is characterised by identifiers and quantifiers and by the position in the n-dimensional network.

Description

Description of the invention

Method for multidimensional analysis of a proteome

The invention relates to a method for multidimensional analysis of a proteome, in which the biological tissue is digested with the proteome to be analyzed and the proteins belonging to the proteome are separated and quantitatively determined and identified. The method is used in biochemistry, in biotechnology, in medicine and in the pharmaceutical industry and serves u. a. for diagnostic purposes and for the development of biologically active substances. Special areas of application open up in basic research, for example to clarify developmental biological or cell-differentiating issues, as well as in applied research for the screening of drug banks, for the development and optimization of biologically active substances or for the differentiation between normal and pathogenic conditions in organisms.

In the recent past, genomes of organisms have been sequenced in whole or in large part [Fraser CM et al .: The minimal gene complement of Mycoplasma genitalium, Science, 1995, Oct 20, 270 (5235), 397-403; Fleischmann RD et al .: Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science, 1995, Jul 28, 269 (5223), 496-512; Blattner FR et al .: The complete genome sequence of Escherichia coli K-12, Science, 1997, Sep 5, 277 5331), 1453-74; Goffeau A et al: Life with 6000 genes, Science, 1996, Oct 25, 274 (5287), 546, 563-7]. CDNA sections were sequenced even more intensely [Clark MS: Comparative genomics: the key to understanding the Human Genome Project, Bioessays, 1999, Feb, 21 (2), 121-30; Evans MJ et al .: Gene trapping and functional genomics, Trends Genet, 1997 Sep, 13 (9), 370-4]. The sequence data are stored in databases. The elucidation of the genome of an organism ultimately "only" leads to knowledge of the relatively static information content of the genetic material for this organism. With the sequences of the cDNA it is possible in principle to determine the expression level of the mRNA also cell-specifically and environmentally-specifically and thus to obtain a gene expression pattern of the RNA. A gene of the genome can a) result from different processes, different types of mRNA which code for divergent proteins, and b) the proteins resulting from them can form a multitude of extraordinarily different functioning proteins through post-translational modification. Modifications known to date include phosphorylation and dephosphorylation, limited proteolysis, acetylation, methylation, adenylation, sulfation, glycosylation [McDonald LJ et al .: Enzymatic and nonenzymatic ADP-ribosylation of cysteine, Mol Cell Biochem, 1994 Sep, 138 (1-2 ), 221-6; Baenziger JU: Protein-specific glycosyltransferases: how and why they do it !, FASEB J, 1994 Oct, 8 (13), 1019-25; Mimnaugh EG et al .: The measurement of ubiquitin and ubiquitinated proteins, Electrophoresis, 1999 Feb. 20 (2), 418-28; Davis PJ et al .: Protein modification by thermal processing, Allergy, 1998, 53 (46 Suppl), 102-5]. However, the expressed and modified proteins ultimately result in the pattern that describes cell differentiation and the reaction to internal and external influences of cells. The most striking is the limited importance of knowledge of the genome for the realization of a defined biological state when one sees the different cells in different organs and within one Organ compares. For example, a liver paranchymal cell, a nerve cell of the brain and a mucosal cell of the intestine have the same set of genetic information, but completely different functions, by regulating the expression of the genome in these cells and regulating the enzyme and protein pattern within the cells as well as the different ones Tissue is caused.

DNA RNA protein

With the exception of the transmission of maintenance, static information. the cell structure, and descriptive amount is regulated in response to vertiv. and transmits the changes and information of the signals. DNA on the interactions with protein level. other cells. Quantity and activity are regulated.

The definition of the "proteome" was not made until 1996 [Friedrich GA: Moving beyond the genome projects, Nat Biotechnol, 1996 Oct, 14 (10), 1234-7].

The proteome, that is, the entirety of all proteins in a cell with a certain level of development and under defined environmental conditions, represents a much more dynamic representation of the physiological state of cells, organs and organisms. The proteome analysis examines which parts of the genome under defined, cell-specific Conditions are expressed and modified. This led to rapidly growing interest in this area, with the consequence of increasing publication numbers (PubMed query search term: proteome; search 1 year ago: 64 entries, 2 years ago: 99 entries, 5 years ago: 122 entries), congresses and events this topic. In order to obtain a quantifiable "image" of a proteome, the current procedure is as follows: In a first step, the biological materials have to be digested and homogenized (with exceptions: for example in the case of serum they are in a homogeneous solution). In the second step the proteins are separated, in the third the identification and in the fourth the evaluation of the data obtained [Ben RH et al .: Two dimensional electrophoresis, The State of the art and future directions, Proteome Research, New frontiers in functionel genomics, Springer 1997 Chap, 2, 13-33].

1. Information

Known methods and arrangements from biochemistry are used for this, such as shear force homogenizers, ultrasound treatment, high-pressure presses. The difficulty lies in a quantitative and, if possible, not damaging the function of the proteins, because only quantitatively digested proteins provide a real picture of the sample material in the subsequent second step (separation and detection of the proteins) [Rabilloud T: Solubilization of proteins in 2-D electrophoresis, An outline, Methods Mol Biol, 1999, 112, 9-19; Rabilloud T et al .: Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients, Electrophoresis, 1997 Mar-Apr, 18 (3-4), 307-16; Staudenmann W et al .: Sample handling for proteome analysis, Electrophoresis, 1998 May, 19 (6), 901-8].

2. Separation and detection

Two-dimensional gel electrophoresis is currently used to separate the proteins of the proteome. First attempts with a two-dimensional HPLC have been made. So far, however, these have not achieved the selectivity of two-dimensional electrophoresis [Opiteck GJ, et al. Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping. Anal biochem. 1998 May 1; 258 (2): 349-61.]. The first dimension of two-dimensional electrophoresis is a separation according to the isoelectric point, i.e. ultimately according to the charge properties of a protein. In the second dimension, the size of the proteins is separated in a denaturing sodium dodecyl sulfate gel. This separation technique has been known for about 20 years. An advantage of 2-D electrophoresis is the ability to separate a relatively large number of proteins on an area with high resolution. It is currently assumed that approximately 10,000 proteins can be detected in such a two-dimensional gel [Klose J et al .: Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome, Electrophoresis, 1995, Jun 16 (6), 1034-59]. Another advantage is that by radioactive labeling or after staining with also known techniques, one is able to quantify the separated proteins. These quantification methods are protein-specific, have a limited dynamic detection range, are generally difficult to automate and depend on the respective (often not fully reproducible) operating conditions [James P: Of genomes and proteomes, Biochem Biophys Res Commun, 1997, Feb 3, 231 (1), 1-6]. They are only suitable for relative determinations. The quantification via immunological properties is problematic because blot techniques with limited quantitative significance have to be used for this.

The result is a fingerprint-like image that characterizes the proteome. The disadvantages of this separation technique are: - Limited dynamic range, which is caused by the resilience of the separating gels

- the maximum amount of protein that can be used is limited to a range from μg to mg protein [James P: Of genomes and proteomes, Biochem Biophys Res Commun, 1997, Feb 3, 231 (1), 1-6]

- Limitation of the sample volume used

- The separation is limited to two dimensions

- The ampholytes required for the separation and the gel material acryl amide can lead to artifacts and thus contribute to misinterpretations that are difficult to identify

Proteins which are present in very high concentrations give relatively strong signals and mask those which are present in low concentrations, so that direct identification and quantification is not possible in this case

- The loss of the native conformation in the denaturing separating gel causes the loss of the biologically functional properties and makes it difficult to identify the proteins by determining their biological properties, such as their catalytic activity or their specific binding properties

- The secondary analysis, like the frequently used, specific proteolysis of individual proteins, followed by mass determination, makes an extraction step from the gel or from the blot membrane that is difficult to automate necessary.

3. Identification of the proteins

For this purpose, sequencing, mass analysis and estimation of the isoelectric point from the running distance in the gel as well as peptide fragment mass analysis after isolation from the gel and tryptic digestion are usually used in mass spectrometry [Shevchenko A et al .: Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels, Proc Natl Acad Sei USA, 1996, Dec 10, 93 (25), 14440-5; Traini M et al .: Towards an automated approach for protein identification in proteome projeets, Electrophoresis, 1998, Aug 19 (11), 1941-9]. Due to the separation technique used, features such as the catalytic activity of the proteins and the native conformation are almost completely switched off and are not available for identification.

The advantages and disadvantages of the known identification methods are in particular:

- The sequencing is done by Edman dismantling in automated facilities and is relatively costly and time consuming. It requires larger amounts of the protein. Therefore, despite current developments, it is less suitable for mass screening [Gooley AA et al .: A role for Edman degradation in proteome studies, Electrophoresis, 1997, Jun 18 (7), 1068-72]. In most cases, this analysis step is necessary to identify primarily unknown proteins.

- The specificity of the statement of the mass determination of a protein, which should ultimately lead to its identification, is increased by subjecting the proteins to a protease digestion after separation and the information obtained by mass analysis with the masses of the peptide sequences predicted from the primary structure after the compares tryptic digestion. Essentially two types of mass spectrometry are used. Firstly, there are matrix-assisted laser desorption ionization - mass spectrometry (MALDI-MS) and secondly, electrospray ionization - mass spectrometry (ESI-MS) [Ducret A et al .: High throughput protein characterization by automated reverse-phase chromatography / electrospray tandem mass spectrometry, Protein Sei, 1998, Mar 7 (3), 706-19; Parker KC et al .: Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination, Electrophoresis, 1998, Aug l9 (ll), 1920-32]. The first method has the advantage that it allows a very large mass range of up to 1 million daltons to be analyzed and can be carried out relatively robustly. However, it can only be carried out batchwise. The ESI technology, on the other hand, can be quasi continuously connected to separation technologies and is currently showing a strong increase both in the development of the range of applications and in terms of technological possibilities. The enormous advances that have been achieved with both techniques in recent years allow mass resolutions up to isotope distribution, i.e. resolutions below 1 Dalton. A mass spectrum of peptide fragments is thus obtained after sequence-specific, defined protease digestion or another defined cleavage of the proteins. This spectrum is typical for every protein and is used for protein identification in sequence databases of proteins and expressed sequence tag banks. Since the identification of the protein comes about through the precise identification of the predicted peptides after protease digestion, any post-translational modification of the proteins, for example by glycosylation, interferes with the recognition. In addition, fragmentation spectra of the individual peptides in the mass spectrometer can provide information about the amino acid sequence of the peptides. This sequence information can be used alone or together with the other known data of the protein to identify it in a sequence database. This method for sequence analysis is currently not in use due to the difficulties of correct data interpretation Routine use. The limits of protein identification using mass spectrometric methods are the incomplete recording of all protein sequences in the existing databases.

4. Data analysis

The characteristics of the individual proteins detected from the separation in 2-D electrophoresis, such as the quantity, isoelectric point and size, and the data for protein identification from further steps, for example sequencing or mass spectrometry, are combined. This gives the picture of the totality of the proteins with their identity and quantity in the respective proteome.

The object of the invention is to improve the quantification and identification of the proteins of a proteome, to facilitate them and to enable them for certain proteins in the first place.

According to the invention, the proteins of the proteome are subjected to a variety of n different separation processes under standardized conditions such that each of the m _t liquid fractions obtained in one separation step in a subsequent separation step yields m ₂ liquid fractions, wherein after n separation steps m _x * m ₂ * .... m _n = M liquid fractions are present, which are identified qualitatively and quantitatively by r different analysis methods by identification methods known per se and quantitatively determined by likewise known quantification methods, so that after the analysis data have been combined, an n-dimensional image of the Proteome, characterized by identifiers and quantifiers as well as by the location in the n-dimensional data space. Advantageous embodiments of the method are listed in subclaims 2-12.

With the method according to the invention, the narrow quantity limitation due to the resilience of the 2-D electrophoresis used to date is no longer present. Amounts of protein in the range of a few grams can be used. The separation matrices can be used several times. This enables a higher reproducibility of the results. The sample material used is in the liquid phase and is therefore immediately accessible to subsequent analysis steps. By better preserving the native properties during the separation, analytical methods such as activity determination and immunological methods based on the native conformation of the analyte are possible. The separation of analytes with the same charge and size properties is not possible in the most commonly used 2-D electrophoresis. However, by using at least one further characteristic, such as the hydrophobicity of the analyte, for the separation, this restriction does not apply. The samples are also available in the fractions for further preparative work after separation.

The invention will now be described with reference to one in the drawing

Embodiment will be explained in more detail.

Show it:

Fig. 1: Separation of 1000 proteins in three dimensions

Fig. La: Fractions 1 to 33

Fig. 2a: Fractions 33/34 to 67

Fig. 3a: Fractions 68 to 100 Fig. 2: Graphical three-dimensional representation of the fractions according to

Fig. 1 As an exemplary embodiment, 1000 proteins are to be described by three properties A, B, C. These properties can be, for example, size, charge and hydrophobicity. The properties are distributed randomly in the proteins. All proteins are numbered consecutively. This is followed by a separation according to property A (for example the size), in which 100 fractions a with the corresponding proteins are obtained. These fractions a are separated into 10 fractions b according to property B (for example the charge).

Each of these fractions b is subjected to a separation according to property C (for example hydrophobicity) and gives fractions c. A total of 100 x 10 x 10 = 10,000 individual fractions are obtained. Each protein obtained by the separation is clearly assigned to one of the fractions a, b, c according to its properties. In the list according to FIG. 1, the respective fractions are designated by numbers. Here, the fractions a belong to property A. They divide the possible value range of property A into one hundred identical parts each, i.e. H. for the assumption of a value range from 0 to 100, for example, the value 1 corresponds to the range 0 to 1, the value 2 to the range 1 to 2, -, the value 100 to the range 99-100. Similarly, the possible value ranges of properties B and C are each divided into ten equal parts, i.e. H. for example, the value 1 corresponds to the range 1-10. On average, every tenth fraction contains a protein. The possibility of multiple occupations results from the random analysis. The example shown in the list according to FIG. La-c contains 39 double assignments and a triple assignment of fractions.

For reasons of space and for the sake of clarity, the empty 9,000 fractions are not shown. 1 contains the following tabular list:

where the fractions a = 1 to 33 are shown in FIG. la, the fractions a = 33/34 to 67 in FIG. 2a and the fractions a = 68 to 100 in FIG. 1c. FIG. 2 shows a three-dimensional diagram with the positions of the fractions occupied by proteins according to FIG. 1.

List of the reference symbols used

A, B, C - property of proteins a, b, c - fraction

Claims

Patent claims

1. Method for the multidimensional analysis of a proteome, in which the biological material is digested with the proteome to be analyzed and the proteins belonging to the proteome are separated and quantitatively determined and identified, characterized in that the proteins of the proteome are a variety of different ones under standardized conditions Separation processes for n>2 are subjected to such that each of the liquid fractions ir^ obtained in a separation step delivers m ₂ liquid fractions in a subsequent separation step, whereby after n separation steps ^ * m ₂ * .... m _n = M liquid Fractions are present which are identified qualitatively and/or quantitatively using r different analysis methods using identification methods known per se and are quantitatively determined using quantification methods also known per se, so that after the analysis data have been combined, an n-dimensional image of the proteome is formed, characterized by identifiers and quantifiers as well as by the location in the n-dimensional data space is obtained.

2. The method according to claim 1, characterized in that, as separation methods, methods that separate according to the size of the proteins, and / or methods that separate according to the mass of the proteins, and / or methods that separate according to the charge of the proteins, and /or methods that separate according to the hydrophobicity of the proteins, and/or methods that separate according to the shape of the proteins and/or methods that separate according to the affinity of the proteins with regard to specific ligands, including antibodies, are selected.

3. The method according to claim 1, characterized in that methods for determining specific immunological properties and / or determining methods of specific catalytic activity and / or determining methods for chemical modification of the proteins of the proteome are used as identification methods.

4. The method according to claim 1, characterized in that methods of non-specific determination of the protein concentration with different sensitivities and / or quantitative determination methods for determining specific catalytic activities and / or quantitative immunological methods and / or quantitative binding assays are selected as quantification methods.

5. The method according to claim 1, characterized in that the identification of individual proteins of the proteome takes place directly by determining the mass of the proteins.

6. The method according to claim 1, characterized in that the identification of individual proteins of the proteome takes place after prolease digestion and mass identification of the fragments.

1. The method according to claim 1, characterized in that in the first separation step the fractions are collected in a two-dimensional multiple vessel system, preferably in the manner and with the base area of microtitration plates.

8. The method according to claim 1, characterized in that in the first separation step the fractions are collected in a defined grid, preferably in the n * 96-fold grid of microtiter plate technology.

9. The method according to claim 1, characterized in that all identification and quantification steps are carried out in a defined grid, preferably the n * 96-fold grid, with suitable liquid handling technology.

10. The method according to claim 9, characterized in that all identification and quantification steps are carried out with at least four pipettor channels arranged two-dimensionally and operating simultaneously.

11. The method according to claim 1, characterized in that the first dimension for separation is a known high-resolution size exclusion, an ion exchange or a hydrophobicity chromatography, that the second dimension by parallel separation and fractionation of the fractions of the first dimension after another as the separation principle used for the first dimension and that any further separation and fractionation is carried out by parallel separation and fractionation methods with the fractions obtained from the previous separation and fractionation steps.

12. The method according to claim 1, characterized in that the analysis data for the n-dimensional image of the proteome are summarized in a database.