WO2003098182A2

WO2003098182A2 - Method for quantifying molecules

Info

Publication number: WO2003098182A2
Application number: PCT/EP2003/005091
Authority: WO
Inventors: Werner Stegmann; Michael Cahill; André SCHRATTENHOLZ
Original assignee: Proteosys Ag
Priority date: 2002-05-15
Filing date: 2003-05-15
Publication date: 2003-11-27
Also published as: AU2003233330A8; AU2003233330A1; WO2003098182A3; WO2003098182B1

Abstract

The invention relates to a method for determining the ratio of the protein frequencies of proteins of the same type in a first and at least one second sample, to a method for determining the concentration of stable isotopes in molecules and to a method for determining the incorporation rate of labelled substances into proteins.

Description

description

Methods for quantifying molecules

The present invention relates to a method for determining the accumulation of stable isotopes in molecules, a method for determining the ratio of the protein frequencies of similar proteins in a first and an at least second sample, a method for determining the rate of incorporation of labeled substances into proteins and a method for Obtaining information about proteins and / or peptides. The samples consist in particular of cells, cell systems, organisms or parts thereof.

In the field of protein analysis, the use of mass spectrometers has experienced a great boom in the past decade. This is related to the development of special mass spectrometric methods with which the masses of large biomolecules, such as proteins or peptides, can be determined with high precision. Based on such mass determinations and information from gene and protein databases, proteins can now be identified in high throughput.

The mass spectrometric methods used are MALDI-TOF mass spectrometry and ESI mass spectrometry. MALDI (matrix-assisted laser desorption ionization) and ESI (electro-spray ionization) are ionization methods with which biomolecules are "gentle", ie. H. non-destructive, can be ionized. For mass detection of the ionized biomolecules

Mass analyzers are used, which can examine ions according to their mass to charge ratio m / z. As Mass analyzers are time of flight (TOF = MS), quadrupole MS, ion trap MS and FTICR (Fourier Transform Ion Cyclotron Resonance) MS (MS = mass spectrometry) in use.

Mass spectra of biomolecules show a mass distribution that arises due to the different masses of the naturally occurring isotopes of the elements from which the biomolecules are built. With mass spectrometers that have a sufficiently large mass resolution, the mass distributions of biomolecules can be resolved into individual mass peaks. Such mass peaks and their distributions are referred to below as isotopomers or isotopomer distributions. Since there is no uniform nomenclature in this area, the term isotopomer is also to be understood as isotopolog or comparable expressions below. The term isotopomer distribution can also be equated with the term isotope pattern.

Examples of the use of mass spectrometry in protein analysis, here in connection with the labeling of analytes with stable isotopes, are the methods described in [Chen 2000], [Veenstra 2000], [Ong 2002] and [Pratt 2002], which determine the determinability improve or facilitate proteins or peptides using mass fingerprints.

In the method described in [Chen 2000], double-deuterated glycine or triple-deuterated methionine is incorporated into proteins of bacteria for qualitative purposes in order to improve the determinability of proteins or peptides by mass fingerprints. Due to the small mass differences between labeled and unlabeled amino acids and the resulting overlap of their isotopomer distributions, this is The method can only be used for peptides with a mass of less than 2000 Da.

In the methods described in [Veenstra 2000] and [Pratt 2002], leucine (Leu-d10) labeled with stable isotopes is incorporated into bacteria. In a similar procedure, in the method described in [Ong 2002], leucine (Leu-d3) labeled with stable isotopes is incorporated into mammalian cell cultures. These studies are designed in such a way that the largest possible mass differences between labeled and unlabeled proteins or peptides are generated in order to separate, non-overlapping isotopomer distributions, which can be determined using MALDI-TOF mass spectrometry or ESI-Q-TOF

Mass spectrometry can be obtained. In addition to the prerequisite that the isotopomer distributions of marked or unmarked molecules are largely free of overlap, a further prerequisite for these methods is that the marked protein or peptide fraction is marked as completely as possible in its marking positions, since only then can quantitative results be achieved. In particular, an accurate quantification of the labeled and unlabeled cells is not possible without a complete incorporation of Leu-d3 in the proteins.

The above-mentioned methods are therefore only suitable for examining organisms or cells in which amino acids with a normal isotope composition can be almost completely replaced by corresponding amino acids labeled with stable isotopes. It is essentially restricted to cells or unicellular organisms that can be grown in culture media labeled with stable isotopes. These conventional methods cannot be used for the "in vivo" labeling of entire organisms, e.g. B. of mice, since the required high levels of marking can not be achieved there. It is therefore desirable to have new methods for obtaining further molecule-specific parameters which enable a further qualitative and / or quantitative analysis of molecules, in particular proteins and / or peptides.

The object of the invention is to provide methods for the qualitative or quantitative analysis of molecules which can be carried out quickly, selectively and inexpensively.

This object is achieved by the subject matter of the independent claims. Preferred embodiments are mentioned in the dependent claims. The wording of all claims is hereby incorporated by reference into the content of the description.

Surprisingly, it was found that statements in this regard can be made by evaluating the isotopomer distribution of molecules.

According to the invention, in a method for determining the enrichment of stable isotopes in molecules, in particular in proteins and / or peptides, the isotopomer distribution of the molecules is determined by mass spectrometry and the enrichment of the stable isotopes is calculated by comparing the determined isotopomer distribution with a reference distribution of the molecules.

In a further development of the invention, the reference distribution is the theoretical, in particular naturally occurring,

Isotopomer distribution of the molecules. Any deviation from the naturally occurring isotopomer distribution can be attributed to the enrichment of the molecules with stable isotopes. Enrichment can be done by labeling the molecules with stable Isotopes occur and / or for example by mixing two samples, one sample having a natural or otherwise known isotopomer distribution and the other sample being labeled with one or more stable isotopes. In the case of a mixture of different samples, the ratio of the protein frequencies of similar proteins in the respective samples can be determined, provided that the isotopomer distributions of the molecules with the corresponding stable isotopes in all samples and all compounds are known and homogeneous.

The isotopomer distribution for this can be determined, for example, by a method for determining the accumulation of stable isotopes in proteins, peptides and / or their fragments. Here, the amino acids and / or the fragments of the proteins and / or peptides are obtained by, for example, hydrolysis and subsequent chromatography. The isotope composition of the amino acids and / or the fragments and / or atoms is determined, and the isotopomer distributions can be determined from the determined isotope compositions. The ICP (Inductively Coupled Plasma) MS is particularly suitable for determining the isotope compositions.

When using this method, advantageously only a mass spectrometry of a mixture of, for example, two different samples has to be carried out in order to be able to determine the ratio of the protein frequencies of similar proteins of these samples.

In a further development of the invention, the molecular composition is known or the molecular composition is determined on the basis of the mass fingerprint of the molecules and the theoretical isotopomer distribution of the molecules is calculated using the Molecular composition and / or with the help of e.g. B. determined a database.

In a further preferred embodiment of the invention, the reference distribution is determined from non-enriched molecules and / or enriched molecules with known enrichment.

Furthermore, the invention comprises a method for determining the different enrichment of stable isotopes in proteins, peptides and / or their fragments from different samples, which is characterized in that the stable isotope enrichment of the pool of one or more individual amino acid constituents in the sample the protein and / or peptide is determined experimentally. Here, amino acids and / or fragments of the proteins and / or peptides are obtained, for example, by hydrolysis and subsequent chromatography or MS / MS or the like, and the isotope composition of the amino acids and / or the fragments is determined. The average enrichment rate of one or more individual amino acid components can be determined from the particular isotope compositions. This

Amino acid enrichment allows a mass spectrometric measurement of the labeled sample without calculating the theoretical isotopomer distributions from a comparison sample.

According to the invention, the method for determining the accumulation of stable isotopes in proteins in a sample comprises the following steps:

Label the proteins of the sample with stable isotopes, - Separation of the proteins of the sample with the help of a

Separation means, in particular a 2D gel, possibly in several steps, optionally obtaining fragments from the separated proteins by specific cleavage or digestion of the proteins before or after the separation,

Determination of the isotopomer distribution of the fragments by MALDI-TOF mass spectrometry and

Calculation of the enrichment of stable isotopes from the determined isotopomer distribution in comparison with an expected isotopomer distribution of a reference value, e.g. B. the naturally occurring or a known isotopomer distribution.

The invention further comprises a method for determining the accumulation of stable isotopes in proteins, peptides and / or their fragments, in which the amino acids and / or the fragments of the proteins and / or peptides are obtained by hydrolysis and subsequent chromatography, the isotope composition of the amino acids and / or the fragments are determined and the enrichment rate is determined from the determined isotope compositions. For example, the ICP (Inductively Coupied Plasma) MS is suitable for determining the isotope compositions.

If more than two samples are examined, for example one sample can remain unlabeled, a second sample can be labeled with, for example, ¹⁵ N and a possible third or further samples with, for example, ¹³ C or equivalent isotopes, and the samples can then be mixed. If the isotope distribution of the amino acids is known for all samples, a mixture of proteins from all samples can be quantified taking into account the isotopomers of several peptides of a protein, in particular by several measurements of differently combined mixtures of the samples. The methods according to the invention have in common that for an analysis of molecules the isotopomer distribution of the molecules is determined with the aid of high-resolution mass spectrometry, in particular MALDI-TOF mass spectrometry. On the basis of the isotopomer distribution determined in this way, it is possible to make various qualitative and / or quantitative statements about the molecules. The isotopomer distributions come about through the existence of heavy isotopes of the elements from which the molecules are built. The greatest influence on isotopomer distributions of peptides is the relatively frequent heavy isotopes of the elements carbon ( ¹³ C = 1.1%) and nitrogen ( ¹⁵ N = 0.37%).

The mass resolution that can be achieved with TOF, in particular MALDI-TOF, mass spectrometry is significantly higher than the mass resolution that can be achieved by other MS techniques (ESI quadrupole or lon-trap MS). Methods with comparable or better mass resolution, such as the FTICR-MS, can also be used.

Surprisingly, it was shown that MALDI-TOF mass spectrometry enables the isotopomer distributions of peptides to be determined reproducibly within narrow error limits.

The reproducibility of such isotopomer distributions, as measured by MALDI-TOF mass spectrometry, has not been thoroughly tested. Although these distributions were often used for a qualitative comparison, quantitative statements were not derived from individual distributions or superimpositions of individual distributions, but only from individual distributions that were widely separated by high isotope concentrations of the molecules. Arguments against their quantitative use were made with falsifying, non-calibratable influences from the MALDI ionization process and the TOF mass analysis.

According to the invention, the method for determining the ratio of the protein frequencies of similar proteins in a first and an at least second sample comprises the following steps:

Label the proteins of at least one sample with stable ones

isotopes

Production of a protein mixture of equal amounts of protein from the first and the at least second sample, separate separation of the proteins of the first sample, the

Protein mixture and the at least second sample with the aid of a separating agent, in particular a 2D gel, optionally in several steps, - optionally obtaining fragments from the separated

Proteins by specific cleavage or digestion of the proteins, before or after the separation or directly after the labeling,

Determine the isotopomer distribution of the fragments by

MALDI-TOF mass spectrometry and - calculation of the ratios of the protein frequencies between the first and the at least second sample from the determined

Isotopomerenverteilungen.

The frequencies of the similar, ie corresponding, proteins in the first or in the at least second sample are unknown. The ratio of these frequencies is determined by the method according to the invention. The method is analogous to determining the ratio of the frequencies of corresponding peptides in the first or in the at least second sample. The proteins of the same type can be present in isolation or, for example, localized in cells. If the proteins are located in cells, the two samples typically consist of similar cell cultures, so-called cell pools. The relative cellular protein frequencies are determined or quantified by the method according to the invention.

In this method, the different isotopomer distributions of the starting samples and the sample mixture or the sample mixtures for calculating the protein frequency ratios and the effects of an influencing agent on these ratios are determined.

In a first step, the proteins of at least one sample are labeled with stable isotopes. Labeling in the sense of the invention means the introduction of elements into the proteins to be labeled which do not naturally occur in this composition or which occur in different relative relationships to one another between the at least two samples. Introducing is understood to mean, for example, the incorporation, particularly in the case of biosynthesis within cells, of labeled amino acids into proteins and peptides to be labeled.

The labeling of cells can advantageously be carried out “in vivo” by growing the cells on a medium which contains labeled amino acids or other sources for the corresponding elements. The amino acids, for example enriched, are absorbed into the cells and incorporated into the cell proteins to be examined. The amino acids can contain, for example, ¹⁵ N, ¹³ C, ^1δ O, ^M S and / or ² H ₂ . Labeling "in vitro" is also possible, for example by protein alkylation.

In a second step, a protein mixture of known amounts of protein from the first and the at least second sample manufactured. An amount of protein includes all of the proteins present in the sample and is determined by conventional methods.

In a further step, the proteins of the first sample, the protein mixture and the at least second sample are separated using a separating agent, in particular a 2D gel. The separation of the first sample and the second sample is only necessary if the accumulation of stable isotopes in these samples is otherwise unknown. This results in a separation of the proteins present in the sample on the basis of specific protein properties. Subsequently, individual proteins found by the separation are analyzed. The frequencies of corresponding, corresponding proteins of the first and the at least second sample are related to one another.

In a next step, the separated proteins are broken down into fragments or peptides by digestion, it being possible for the fragmentation to have been carried out beforehand.

The isotopomer distribution of the fragments is subsequently determined by MALDI-TOF mass spectrometry. The

Isotopomer distributions of the fragments can be determined in high resolution, reproducibly within narrow error limits. This is a basic requirement of the procedure.

In a last step, the ratios of the protein frequencies between the first and the at least second sample are calculated from the determined isotopomer distributions. The calculation can be carried out using the isotopomer distributions of a corresponding fragment from the first and the at least second sample. The calculation can also be carried out using the isotopomer distributions of the otherwise known or estimated Stable isotope enrichments of each individual sample take place. Several isotopomer distributions of different fragments or peptides of a protein can also be used for the calculation.

The illustrated sequences of the steps of the method according to the invention are selected only as examples and are not mandatory.

The method described in [Vogt 1993] for the determination of isotope enrichments using

For example, isotopomer distributions can be used directly to calculate the ratios of protein frequencies. This makes use of the fact that the natural isotopomer distribution of a molecule is specifically shifted when the molecule is artificially enriched with isotopes that do not occur in this frequency in nature.

The calculation is advantageously based on the determination of the relative frequencies of peaks in the isotopomer distribution of the proteins or peptides. These peaks are designated with integer values (i = 0, ..., n), which describe the mass offset with respect to the monoisotopic peak (i = 0) in the direction of increasing m / z values. The monoisotopic peaks of the protein or peptide molecules are the peaks with the lowest m / z values in an isotopomer distribution. The isotopomer distributions of molecules are mass spectrometric images of their isotopological distribution Mi (i = 1..n), where Mi denotes isotopologists with the same mass offset i.

So-called RIA values (Relative Isotopologue Abundance values) are used as the calculation variables, which determine the relative number of all isotopologists with the same mass offset i of a protein or Represent peptides based on the sum of all mass spectrometrically determinable isotopologues of the protein or peptide. Accordingly, an RIA value of an isotopologist Mi of a specific protein or peptide fragment p results from:

where S _p, i (i = 0, ..., n) are the ion signals measured by mass spectrometry, which are derived from an isotopomer distribution of the n peaks of an isotopological distribution of a peptide p. The ion signals are determined from integrals via the corresponding peaks.

If the molar amount of an isotopically labeled protein is denoted by L and the molar amount of a protein in a naturally isotopic composition is denoted by N, the mole fraction C of a mixture of these proteins is given by:

C = IJ (L + N) (2)

The mole fraction C is determined according to [Vogt 1993]:

_ RIA _{p ι} (L + N) - RIA _{p ι} (N) RIA _pj (L) - RIA _{p ι} (N)

By inserting the measured values RIA _p , j (L + N), RIA _pi (L) and RIAp j (N), the corresponding mole fraction C _Pι j can be calculated, RIA _p, i (L + N) being the RIA values of the mixture, RIA _Pιi (L) are the RIA values of the labeled and RIA _Pι j (N) are the RIA values of the naturally occurring proteins or peptides. The molar ratio R = UN results from:

(4)

N 1 - C

All n peaks of the isotopomer distribution can be used to determine the mole fraction C _p or the molar ratio. So you get an n-fold redundancy, which can be used to increase the accuracy.

The use of isotopomer distributions, which can be determined with high resolution and reproducibly, for example with the help of MALDI-TOF mass spectrometry, to calculate ratios of protein frequencies, for example with the aid of the method described in [Vogt 1993], enables the determination of ratios of protein frequencies (e.g. B. quantification) or mixing ratios with low isotope enrichments, for example, about 20%. All previously published methods rely on achieving very high isotope accumulations (> 90%) in one labeling step in order to generate separate, overlay-free and defined isotopomer distributions for labeled and unlabeled fragments or peptides and as complete a labeling of all designated labeling sites as possible [Gygi et al. , 1999 and other groups].

The process described here is many times faster and cheaper than comparable conventional processes in which isotope enrichments above 90% are required. It is therefore a new process that offers considerable advantages over the previously published processes. The method according to the invention has the advantage that it does not require knowledge of how the label gets into the peptide and / or protein and whether it is one or more identical or different labels. In addition, it does not matter to what extent the label is enriched in the proteins or peptides. The only decisive factor is that the peptide fragment measurements measured by mass spectrometry

Differentiate the isotopomer distributions of the labeled and unlabeled proteins to be compared sufficiently to be evaluable.

In an advantageous development of the invention, at least one sample is influenced, in particular stimulated, with at least one influencing agent before the production of the protein mixture. The sample advantageously represents a biological sample, for example a cell culture or a cell system. The sample can also be an organism or a part thereof or can be derived from it. The ratios of the protein frequencies are a measure of the effect of the influencing or stimulation on the protein frequencies. In this way, for example, the effectiveness of an influencing agent on cell growth can be examined. The same can be done with inhibition.

The invention further comprises a method for determining the rate of incorporation of substances labeled with stable isotopes, in particular amino acids, into proteins which are formed by cells, with the following steps:

Mixing the labeled substances with the cells, obtaining at least one protein harvest by removing the proteins at at least one specific point in time - separate separation of the proteins from the respective protein crops

Using a separating agent, in particular a 2D gel, possibly in several steps, optionally obtaining fragments of the separated proteins of the respective protein harvests by specific cleavage or digestion, in particular tryptic digestion, of the proteins before the after the separation, determining the isotopomer distribution of the fragments of the respective protein harvests by MALDI-TOF mass spectrometry and

Calculation of the incorporation rate of the substance from the determined isotopomer distributions of the respective protein crops.

Alternatively, the degree of enrichment can be determined from a measurement if deviations from the naturally occurring or otherwise expected isotopomer distribution are observed. Different levels of enrichment in different proteins indicate different incorporation rates.

In the method according to the invention, the incorporation rate of substances, for example amino acids, which are labeled with stable isotopes, into proteins or peptides is calculated. The incorporation rate is understood to mean the change in the amount of substance incorporated in a protein per unit of time. The substances can be labeled amino acids, for example. The labeled amino acids can be introduced into a cell sample or a cell pool, the cells of the sample from the time of introduction of the substance, ie. H. of the labeled amino acids, begin to incorporate the substance into the cell's own proteins or peptides.

In a first step of the method according to the invention, the labeled substances are mixed with the proteins. From this point in time, the substance can be incorporated into the proteins at a specific rate. In a further step, at least one protein harvest is obtained by removing the proteins at at least one specific point in time. As time progresses, substances are increasingly incorporated into the proteins according to the rate of incorporation. If protein harvests or protein samples are taken at defined times, it is possible to determine the incorporation rate on the basis of these protein harvests. If the sample to be examined is a labeled cell sample, cells are removed from the sample, washed and lysed.

In a next step, the proteins of the respective protein crops are separated using a separating agent, in particular a 2D gel. This results in a separation of the proteins present in the sample on the basis of specific protein properties. Subsequently, individual proteins found by the separation are analyzed. The concentrations of the substances in the corresponding, corresponding proteins of the successive protein harvests are related to one another.

Subsequently, fragments of the separated proteins of the respective protein crops are obtained by specific cleavage or digestion, in particular by tryptic digestion, of the proteins.

The isotopomer distribution of the fragments of the respective protein crops is then determined by MALDI-TOF mass spectrometry. The isotopomer distributions of the fragments can be determined in high resolution, reproducible within narrow error limits. This is a basic prerequisite of the method, since the enrichment of the protein in two successive points in time, depending on the installation rate and the time difference between the different points in time, can differ only slightly. In a final step, the rate of incorporation of the substance is calculated from the isotopomer distributions of the respective protein crops. If the peptide sequence is known and with it the amino acids present, the incorporation rate for specific amino acids can be calculated.

Isotopomer distributions can, for example, be used directly to calculate the rate of incorporation of the substance from the determined isotopomer distributions of the respective protein crops.

In a preferred embodiment, the method for determining the incorporation rates of labeled substances in proteins is used to determine the effect of an influence, in particular stimulation, on the incorporation rates of similar proteins in a first and an at least second labeled sample. At least one of the samples is influenced with an influencing agent. The installation rates of the substance are determined for the respective sample and the effect of the influence is determined by forming the ratio of the respective installation rates.

In a preferred embodiment, the proteins are labeled by amino acids which have been enriched with stable isotopes. Here, for example, a cell culture is grown on a medium which contains amino acids enriched with isotopes. This can be the same or different amino acids with the same or different isotope labels.

In further preferred embodiments, the isotopes are ¹⁵ N, ¹⁴ C,

13 13 _N 18Q 34g _{and or} 2 _H2 In particularly preferred embodiments of the methods according to the invention, the isotope enrichments are less than 95%, preferably less than 90%, in particular less than 80%, preferably less than 40% and particularly preferably less than 30%. Known methods are dependent on high isotope enrichments of, for example, greater than 90%, since, for example, a superposition of the isotopomer distributions of the naturally occurring molecules with the isotopomer distributions of the enriched molecules makes analysis difficult or impossible. The combination of MALDI-TOF mass spectrometry in connection with, for example, the method described in [Vogt 1993] makes it possible for the first time to evaluate such low isotope accumulations. This fact is also an important advantage when determining installation rates, since only slight differences in the isotope accumulations can occur between two different measurement times.

The invention further comprises a method for obtaining information about amino acid sequences of peptides, in which the information about the amino acid sequences is determined by means of high-resolution mass spectrometry

Isotopomer distribution of the peptides is obtained.

In the method according to the invention, the isotopomer distribution of a peptide or protein is used to obtain information about amino acid sequences of the peptide in addition to other specific parameters, for example the peptide mass. This information can be used as additional information in the qualification of the peptide to be examined. For example, in a peptide in the peptide mass fingerprint method next to the m / z value the isotopomer distribution can be used as an additional independent search parameter.

In a development of the method, the information about the amino acid sequences relates to the amino acid sequence composition of the peptides. Stabilisotope enriched peptides must contain stabilized isotope enriched amino acids. If the distribution of the stable isotopes over all amino acids is known, the isotopomer distribution of the peptides of a protein also contains amino acid sequence information, because the stable isotope incorporation is sequence-specific. This applies not only to artificially enriched stable isotopes, but also to naturally occurring stable isotopes, especially in the presence of sulfur-containing amino acids.

The presence of the sulfur-containing amino acids cysteine and methionine in a peptide leads to a specific isotopomer distribution of such peptides due to the characteristic isotope distribution of the element sulfur. The element sulfur has the 4 stable isotopes ³² S (frequency 95%), ³³ S (0.75%), ^ S (4.2%) and ³⁶ S (0.015%). The high proportion of the heavy isotope ³⁴ S of 4.2% shifts the focus of the isotopomer distributions of cys- or met-containing peptides towards heavier masses. If ^ S is artificially enriched, this effect becomes greater.

If the isotopomer distribution of peptides is determined, the detection of the shift in the center of gravity of the isotopomer distribution towards heavier masses is a feature of the presence of stable isotope-containing, in particular sulfur-containing, amino acids, according to a further preferred embodiment of the invention. In a further development, the invention comprises a method for improving the ionization ability of peptides and / or proteins after an enzymatic and / or chemical digestion. After digestion, various amino acids are formed both at the C- and N-terminal ends, which vary greatly in their ionization capacity due to their different pK values. In the case of tryptic digestion, for example, the lysine peptides are severely disadvantaged compared to the arginine peptides. By modifying the, preferably N- and C-terminal, amino acids, especially in the region of the side chains, with a first modification substance, the ionization efficiency of, in particular lysine-containing, peptides is increased and the strength of the signals of the mass spectrometric analysis is greatly increased. As such substances, for example, O-methylisourea or nicotinyl-N-hydroxysuccinimide [James 2000] are used for both C- and N-terminal modifications.

The molecules to be examined, in particular proteins and / or peptides, are first cleaned before the mass spectrometric examination and separated, for example with the aid of a polyacrylamide gel. A high stoichiometric efficiency is obtained for the modification of amino acids by immobilizing the proteins or peptides, in particular in a polyacrylamide gel matrix or on a reversed phase material.

The invention further comprises a method for identifying molecules which contain at least one phospho group or carbohydrates linked via an oxygen atom, in particular peptides and / or proteins, such as, for example, phosphoserines or phosphothreonines. The process removes sulfur-containing groups, preferably by Michael addition of sulfur-containing nucleophiles or by beta-elimination of at least one Phospho group, in particular by nucleophilic sulfur-containing reagents, which displace at least one phospho group, introduced. Subsequent detection of the shift in the center of gravity of the isotopomer distribution to heavy masses, due to the displacement of the phospho group by the sulfur-containing nucleophile, is indirect evidence of the presence of a phosphoprotein or peptide.

In a continuation of the process, the introduced sulfur-containing group, in particular a sulfhydryl group, is replaced by a more easily ionizable group in the form of a suitable reagent, preferably an alkylating agent, in particular 3- (acrylamidopropyl) trimethylammonium chloride [Brune 1992] or 1, 1, 3 , 3-tetramethylguanidine, thereby replacing and simplifying the identification of phospho group-containing peptides and proteins, in particular by increasing the strength of the signals in mass spectrometric detection and improving the statistics for higher isotopomers.

The negative charge, for example of a phosphate group, is thereby converted to a basic group. In a further development of the method, in which the Michael addition takes place before, for example, tryptic digestion, new tπ / ptic cleavage sites can be introduced. The combination of new cleavage sites and thus new peptides, together with an isotopomeric sulfur-specific signature, clearly identifies the presence and position of the phosphate group according to the invention. If the Michael addition is carried out with a mixture of reagents, at least one reagent introducing a positive charge and at least one of these reagents introducing no positive charge, then two peptides (= one pair of peptides) are formed per phosphate with mass differences which are dependent on that mass difference the at least two different alkylation molecules: (1) from tryptic cleavage after the amino acid containing the phosphate, (2) from tryptic cleavage after the closest basic amino acid. For all phosphate-containing peptides, these peptide pairs have an isotopomeric sulfur-specific signature, which according to the invention can be attributed to the reagent introduced during the Michael addition.

The invention relates to the analysis of samples from proteins or mixtures of proteins or fragmented proteins, in which the isotopomer distributions can be measured with a sufficiently large mass resolution.

In the already known mass analysis of isotopomer distributions (MIDA), for example described in Analytical biochemistry 267, 1-16 (1999), the fate of a specific precursor, for example labeled leucine, is followed. In contrast to the method according to the invention, it is not possible with MIDA to use more than one labeled precursor, for example a labeled amino acid. In contrast, the methods according to the invention have the further advantage that the labeled precursor (s) can also be metabolized into other amino acids. The methods disclosed here using RIA can be used with more than one precursor. By comparing observed and expected isotopomer distributions, they can be performed on a sample. In addition, qualitative and / or quantitative statements about proteins from two or more samples can be made with these methods, whereby for quantitative statements protein samples are preferably mixed with one another and a mass spectrum of this mixture is analyzed. It is irrelevant what type the stable isotope (s) are, which are incorporated into the peptides and / or proteins, or how the label is metabolically incorporated or converted. This makes it possible, for example, to use inexpensive stable isotope sources, such as, for example, yeast extracts, which are labeled with ¹⁵ N, the amino acids for example being partially or completely labeled. Such isotope sources are suitable, for example, for feeding mice or other experimental animals.

The different isotope patterns of the ions from different samples are determined empirically, which makes it possible to (a) determine the differences between two samples by measuring the relative isotopomer abundances of each sample and (b) the mole fraction of each sample, i.e. its Quantify relationship to each other by using the mixture of two samples for mass spectrometric measurement.

Proteins or peptides which can be investigated according to the invention consist of molecules with two or more amino acids which are linked by one or more peptide bonds with the aid of a ribosome. For the purposes of the invention, synthetic or artificially synthesized polypeptides or proteins can also be produced as long as they are also synthesized by ribosomes. For example, translation systems based on cell extracts and thus producing proteins can also be used.

The invention further comprises a method in which at least two differently labeled samples are analyzed. The samples preferably originate from complex biological systems, such as, for example, from cultivated cells, organisms, tissues Organisms, cellular fractions or chromatographic fractions from biological systems. These samples generally do not consist of individual proteins, the chemical composition of which would be known.

An important aspect of the invention is to increase the sensitivity of existing methods in order to enable the analysis of different proteins in proteome applications. For example, the response of a system, in particular a complex biological system, to experimental treatments can be examined. The required sensitivity of the method according to the invention results u. a. from the examples in combination with the figures.

The samples generally contain various proteins and / or protein isoforms. These protein isoforms are chemically different types of protein that are synthesized by one or more mRNA molecules, the mRNA molecules being transcribed by the same or at least by highly homologous genes. For example, protein isoforms include proteins that are synthesized by differently spliced mRNAs or proteins that are modified differently post-translationally. The proteins or protein isoforms used according to the invention are synthesized on ribosomes and are not synthetically produced by non-biological, chemical methods. In the event that synthetically synthesized polypeptides or proteins of this type are added to the samples, the invention covers methods in which individual protein abundances of non-synthetically produced polypeptides or proteins are determined in at least one sample.

In contrast to the known mass spectrometric analysis of isotopomer distributions (MIDA), it is advantageous for that The method according to the invention does not need to know the amino acid sequence of the peptides or proteins to be analyzed. Rather, the method according to the invention is suitable for analyzing and in particular quantifying previously completely unknown peptides, for example containing cysteine. An aim of the invention is to detect those proteins or peptides in the samples which have different stable isotope contents and / or to differentiate these proteins in different samples with regard to their relative abundance or frequency, in order to thus quantify the differences in to enable the frequencies. The method according to the invention makes it possible to provide information about relative changes in the frequencies of a single protein or a group of proteins between different samples and the changes in the proportional stable isotope content of these proteins, as results, for example, from different experimental treatment of the biological systems that the source can provide for the samples that are analyzed according to the invention.

The different samples can initially be identical. On the other hand, the origin of the samples can be experimentally equivalent categories of the sample, such as different animals or groups of animals or different cultures of cultured cells derived from a mother culture or groups of a mother culture. Before the cells are harvested or removed, the different samples, in particular the different cell cultures or animals or groups of animals, are treated differently during the course of the experiment. For control samples, the experimental conditions should be chosen accordingly. With the method according to the invention, the relative stable isotope contents can be determined and thus the relative quantitative relationships between different multiple pairs or groups of polypeptide ions or protein ions of the same chemical structure or the same monoisotopic mass can be determined from the different samples. Said ions differ only in their stable isotope content within the different samples. One or more daughter ions, such as tryptic peptide ions, from the same protein in each sample can be used to calculate the differences in stable isotope content and / or in the protein frequency ratio between the samples.

The proteins and / or peptides in the samples can be subjected to different labels with stable isotopes by metabolically labeling the living systems, in particular cells, before, during or after the treatment. The labeling reagents do not have to be homogeneous. The samples can additionally or alternatively be labeled with different stable isotopes, for example by adding alkylation reagents with different stable isotope contents to the proteins or peptides of each sample. These alkylation reagents need not have homogeneous stable isotope compounds. Various stable isotopic elements can be combined in a sample for labeling, for example ² H, ¹³ C and / or ¹⁵ N. These different isotopic elements can be incorporated into the different chemical compounds of the proteins and / or peptides.

According to the method according to the invention, the actual or alleged amino acid sequence of the analyzed ions can be identified, for example, by peptide mass fingerprinting or by measuring the masses of the fragments of the analyzed ions. Furthermore can the distribution of the stable isotopes over certain amino acids can also be determined by computer simulation.

In a preferred embodiment, the ions are fragmented during mass spectrometry in order to generate daughter ions which differ in their masses from one another due to components which are linked to the ion via peptide bonds, such as amino acids, post-translationally modified amino acids or other chemically modified amino acids. For example, a fragmentation is carried out for this, which is based on a collision in MS / MS analyzes or is based on a "post source decay fragmentation" after MALDI ionization. An analysis of the isotopomer distributions of the daughter ions enables a determination of the stable ones Isotope levels of each amino acid in the polypeptide ions from each individual sample.

Advantageously, one or more changes in the stable isotope content of the proteins or peptides can be used in combination with information from other sources to interpret the response of the biological system to the experimental conditions. For example, the possibly observed higher frequency of stable isotopes in one or more proteins known to be involved in a stress response may indicate an opposite effect or a toxic response to a particular experimental treatment for certain cells in the system. Furthermore, for example, a change in the content of the stable isotopes in one or more proteins, which presumably play a role in redox regulation, can indicate an oxidative stress or hydroxyl reaction of the system to the treatment. In addition to the method according to the invention with regard to quantitative statements, this aspect of the invention also relates to the method according to the invention with regard to qualitative statements. For the method according to the invention with regard to quantitative statements, the results regarding the abundance of one or more proteins in combination with information from other sources are evaluated in order to interpret the response of the biological system to the experimental conditions.

The method according to the invention is particularly suitable for examining two or more proteins or peptides in one or more samples. Advantageously, for example, even more than 10 or even more than 100 different proteins can be analyzed in one or more samples according to the invention.

In a preferred embodiment of the invention, the method according to the invention is carried out in an automated manner, for example using robots or the like. This automated procedure can be used, for example, for the preparation of the samples, the separation of the samples, the fragmentation of the samples and / or for carrying out the mass spectrometric analysis, which advantageously increases the sample throughput and thus of course generally reduces the costs.

The mentioned features and further features of the invention result from the following description of experiments in connection with the subclaims and figures. The individual features can be implemented individually or in combination with one another.

The pictures show: Fig. 1: Measured with MALDI-TOF mass spectrometry

Isotopomer distribution of a peptide with a mass of approx. 840 DA,

Fig. 2: Isotopomer distribution of a peptide with a mass of approx. 1,570 DA measured with MALDI-TOF mass spectrometry,

Fig. 3: Isotopomer distribution of a peptide with a mass of approx. 2,200 DA measured with MALDI-TOF mass spectrometry,

Fig. 4: Measured with MALDI-TOF mass spectrometry

Isotopomer distribution of a peptide with a mass of approx. 3,300 DA,

Fig. 5 and 6:

The figures show peptides from different mass ranges of the mass spectra belonging to the same protein from a cell pool A, a cell pool B and from a mixture AB.

methods

The steps or methods used in the following experiments are designated with the help of letters, to which reference is made below.

a) is a published method: Oda et al., 1999; Pasa-Tolic et al., 1999; Smith et al., 2001.

b), c), d) and e1) are 'state of art' methods: Kellner, Lottsspeich, Meyer, Microcharacterization of Proteins, Wiley-VCH, 1999; Schrattenholz (ed.), Methods of Proteome Research, Spektrum Verlag, 2001.

e2) is the basic requirement for the procedures described here. This basic requirement was found and verified by our own measurements.

f) is a published method: Vogt et al., 1993.

The application of f) to e), ie the use of the isotopomer distributions of MALDI-TOF spectra for the calculation of mixing ratios of normal and weakly ¹⁵ N (or ¹³ C) -enriched proteins, was also found and confirmed by our own measurements.

An Autoflex 3 from Bruker Daltonics was used as the mass spectrometer. Experiment 1:

Determination of mass spectra of peptides using MALDI-TOF mass spectrometry.

The mass spectra of peptides with masses of z. 840, 1,570, 2,200 and 3,300, for example, show the typical isotopomer distributions shown in Fig. 1 to Fig. 4. These distributions result from the existence of heavy isotopes of the elements from which the peptides are constructed. The relatively common heavy isotopes of the elements carbon (C ¹³ = 1.1%) and nitrogen (N ¹⁵ = 0.37%) have the greatest influence.

Experiment 2: Reproducibility of those measured with MALDI-TOF

Isotopomerenverteilungen.

The enzyme trypsin is used to digest the separated proteins. Trypsin not only digests the proteins but also itself, i.e. H. specific trypsin fragments arise with each digestion. These trypsin fragments have defined masses (e.g. 842.5, 2.211, 2.283, 1.045) and are used in MALDI-TOF spectra of tryptically digested proteins to calibrate the mass scale. They are therefore particularly suitable for determining the reproducibility of isotopomer distributions using MALDI-TOF.

The agreement or reproducibility of 100 isotopomer distributions of the trypsin fragments of the masses 842.5, 2,211, 2,283, 1,045 was tested. The distributions agree with each other within a fluctuation of ± 5%. Within this error, the mean distributions also agree with the theoretical isotopomer distributions of the trypsin fragments. In addition to trypsin fragments, the method was also verified for BSA fragments.

Experiment 3:

Isotopomer distributions of peptides from MALDI-TOF mass spectra contain information about the protein sequence of the peptides.

The isotopomer distributions, which are caused by the presence of the sulfur-containing amino acids cysteine and methionine, were investigated.

The element sulfur has the 4 stable isotopes ³² S (frequency 95%), ³³ S (0.75%), ^M S (4.2%) and ³⁶ S (0.015%). The high proportion of the heavy isotope ³⁴ S of 4.2% shifts the focus of the isotopomer distributions of C- or M-containing peptides towards heavier masses.

The results show that the isotopomer distribution shifts caused by heavy sulfur isotopes can always be clearly demonstrated with MALDI-TOF if the C- or M-containing peptide can be measured without overlap with sufficiently high resolution.

Experiment 4:

Relative quantification of cellular protein frequencies with the help of stable isotopes (2-pool method).

Cell pool A: labeled cells Cell pool B: unlabeled cells a) The cells from cell pool A are grown in a medium which

Contains ¹⁵ N (or ¹³ C) enriched amino acids. The ¹⁵ N-enriched amino acids are taken up by the cells and into the

Cell proteins incorporated. b) stimulation of the unlabeled cells from cell pool B. c) mixture AB: mixture of equal amounts of protein from cell pool A and

Cell pool B. d1) The separation of the proteins from cell pool A, cell pool B and mixture

AB is done using 2D gel electrophoresis. d2) In-gel tryptic digestion of the separated proteins. e1) Measurement of the ¹⁵ N (or ¹³ C) accumulations of the proteins

Peptides obtained from the tryptic digestion of the proteins (MALDI-TOF method). The results are the m / z

Values and the isotopomer distributions of the peptides. e2) With MALDI-TOF, the isotopomer distributions of the peptides are demonstrated very reproducibly. f) From the isotopomer distributions of corresponding tryptic ones

Protein fragments from cell pool A, cell pool B and mixture AB are used to calculate the protein mixture ratios PM. Mixing ratios PM> 1 and PM <1 are a measure of the effect of the stimulation on the protein frequencies.

The measuring range P = 0.1 to PM = 10 can still with an error <± 10

% can be determined.

Experiment 5:

Relative quantification of cellular protein frequencies using stable isotopes (3-pool method).

Cell pool A: labeled cells Cell pool B: unlabeled cells Cell pool C: unlabeled cells a) The cells from cell pool A are grown in a medium which contains ¹⁵ N (or ¹³ C) -enriched amino acids. The ¹⁵ N-enriched amino acids are taken up by the cells and incorporated into the cell proteins. A larger amount of cell pool A is grown to perform multiple stimulation experiments. This amount can also be used as a reference in further experiments and / or to produce a test set. b) stimulation of the unlabeled cells from cell pool B. d) mixture AB: mixture of equal amounts of protein from cell pool A and cell pool B. c2) mixture AC: mixture of equal amounts of protein from cell pool A and cell pool C. d1) separation of the proteins from cell pool A, Cell pool B, cell pool C, mixture AB and mixture AC are carried out by means of 2D gel electrophoresis. d2) In-gel tryptic digestion of the separated proteins. e1) Measurement of the ¹⁵ N (or ¹³ C) enrichment of the proteins in peptides which are obtained from the tryptic digestion of the proteins (MALDI-TOF method). The results are the m / z values and the isotopomer distributions of the peptides. e2) With MALDI-TOF, the isotopomer distributions of the peptides are demonstrated very reproducibly. f) The protein mixture ratios PM are calculated from the isotopomer distributions of corresponding tryptic protein fragments from cell pool A, cell pool B, cell pool C, mixture AB and mixture AC. Mixing ratios P _M > 1 and P _M <1 are a measure of the effect of stimulation on protein frequencies.

The measuring range P _M = 0.1 to P = 10 can still be determined with an error <± 10%. Experiment 6:

Quantification of cellular protein turnover rates or incorporation rates with the help of stable isotopes.

Cell pool A: labeled cells

Cell pool B: labeled stimulated cells a) The cells from cell pool A are placed at a time to in a medium which contains ¹⁵ N (or ¹³ C) -enriched amino acids. The ¹⁵ N-enriched amino acids are absorbed by the cells from time to and incorporated into the cell proteins. b) The cells from cell pool B are placed at a time to in a medium which contains the stimulating substance and ¹⁵ N (or ¹³ C) -enriched amino acids. The ¹⁵ N-enriched amino acids are absorbed by the cells from time to and incorporated into the cell proteins. c) Cell harvesting: A _t0 , An, At2, A _t3 , ... and B _t0 , B _t ι, Bß, B _t3 , ...

At certain times ti, t ₂ , t ₃ , ...> t ₀ , the cells from the

Culture media removed, washed and lysed. d1) Protein separation using 2D gel electrophoresis. d2) In-gel tryptic digestion of the separated proteins. e1) Measurement of the ¹⁵ N (or ¹³ C) enrichment of the proteins in peptides which are obtained from the tryptic digestion of the proteins (MALDI-TOF method). The results are the m / z values and the isotopomer distributions of the peptides. e2) With MALDI-TOF, the isotopomer distributions of the peptides are demonstrated very reproducibly. f1) From the isotopomer distributions of corresponding tryptic protein fragments from the cell harvests A _t0 , A _t ι, As, A _t3 , ... the normal incorporation rate E _N = Δ ¹⁵ N / Δt of the stable isotope ¹⁵ N (or E _N = Δ ¹³ C / Δt for ¹³ C) calculated in the proteins. f2) From the isotopomer distributions of the tryptic protein fragments from the cell harvests B _t o, B _t ι, B _t2 , B _t3 , ... the incorporation rate is below Stimulation E _s = Δ ¹⁵ N / Δt of the stable isotope ¹⁵ N (or E _s = Δ ¹³ C / Δt for ¹³ C) in the proteins was calculated. f3) The effect of stimulation on the protein turnover rate is calculated from the comparison of the installation rates E _N versus.

Experiment 7:

Relative quantification of the protein frequencies of murine stem cells with the help of the stable isotope ¹⁵ N.

The experiment was carried out on murine stem cells. The designation of the individual steps is made according to the previous experiments. In step a), the proteins from cell pool A were enriched to about 25% in ¹⁵ N (based on the natural ¹⁵ N isotope frequency 0.37%). Cell pool B consisted of stem cells with a natural isotope composition. After carrying out steps b) - d), the MALDI spectra of the tryptically digested proteins from cell pool A, cell pool B and mixture AB were measured in e1) and e2). A result example is shown in Figs. 5 and 6. The pictures show peptides from different mass ranges of the mass spectra belonging to the same protein from cell pool A, cell pool B and from the mixture AB. A mixture ratio of 1.1: 1 with a standard deviation of ± 6% for the mixture proportions of cell pool A and cell pool B in the mixture AB was calculated from the MALDI spectra from FIGS. 5 and 6 using f). Within the error limits, this confirms what has been experimentally set due to pipetting

Mixing ratio of 1: 1. Experiment 8:

Examination of brain proteins in mice.

The method according to the invention allows the detection of very small amounts of stable isotopes in proteins, which enables the following experiment.

Mice are fed stable isotope-labeled amino acids. In principle, the diet can contain one or more amino acids of the 20 occurring amino acids. The labeled amino acid can advantageously be 99% enriched with the stable labeled amino acid. In this experiment, D10 leucine is used, which is only minimally metabolized into other products and induces a mass shift of 10 per leucine.

The amino acid pool that is available for protein synthesis in the mouse is fed by the material taken in with the feed and the breakdown of existing proteins into amino acids. Since the biomass of the proteins in the mouse is large, incorporation of more than 90% into new proteins is difficult to achieve.

The newly synthesized proteins are chemically indistinguishable from existing proteins that do not contain labeled stable isotopes. Therefore, the stable isotopes are diluted in the amino acid pool stage and in the mixture stage with the existing proteins.

After starting the diet with the stable isotopes, the mouse is stimulated. For this, the mouse is subjected to a tutorial. The learning process is associated with the synthesis of new proteins in certain regions of the world Brain, especially the hypocampus. Therefore, a tutorial is to be equated with the influencing agent as described in the general part above.

After the learning process of the mice fed with the labeled amino acids, the hypocampus is extracted and the various proteins are separated using a 2D PAGE. The proteins are then fragmented. The analysis is done for proteins that contain more stable isotopes in the mice of the tutorial than in the mice without a tutorial. Detection of increased stable isotopes in a particular protein indicates translational up-regulation of the protein in response to the stimulus, that is, to learning.

Assume the increase for a particular protein is 10% and this newly synthesized protein has 50% labeling efficiency. It follows that the rise of the stable isotope in the pool of this protein is 5%. Under these circumstances, neither 2D PAGE nor conventional mass spectrometry quantification is able to detect this change. In contrast, the method according to the invention can identify this protein which is involved in the response to the stimulus.

literature

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Gygi S.P., Rist B., Gerber S.A., Turecek F., Gelb M.H., Aebersold R .; Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotech., 77, 994-999, 1999.

Kellner, Lottspeicher, Meyer; Microcharacterization of proteins; Wiley-VCH, Weinheim, 1999.

Mann M .; Quantitative proteomics? Nature Biotechnology, 17, 954-955, 1999.

Oda Y., Huang K., Cross F.R., Cowbum D., Chait B.T .; Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Be. USA, 96, 6,591-6,596, 1999.

Pasa-Tolic L., Jensen P.K., Anderson G.A., Lipton M.S., Peden K.K., Martinovic S., Tolic N., Bruce J.E., Smith R.D .; High throughput proteome-wide precision measurements of protein expression using mass spectrometry. J. Am. Chem. Soc, 121, 7.949-7.950, 1999.

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Smith R.D., Pasa-Tolic L., Lipton M.S., Jensen P.K., Anderson G.A., Shen Y., Conrads T.P., Udseth H.R., Harkewicz R., Belov M.E., Masseion C, Veenstra T.D .; Rapid quantitative measurements of proteomes by fourier transform ion cyclotron resonance mass spectrometry. Electrophoresis, 22, 1,652-1,668, 2001.

Veenstra T.D., Martinovic S., Anderson G.A., Pasa-Tolic L. and Smith R.D .; Proteome analysis using selective incorporation of isotopically labeled amino acids. J. Am. Soc. Mass Spectrom., 11, 78-82, 2000.

Vogt J.A., Chapman T.E., Wagner D.A., Young V.R. & Burke J.F .; Determination of the isotope enrichment of one or a mixture of two stable labeled tracers of the same compound using the complete isotopomer distribution of an ion fragment; theory and application to in vivo human tracer studies. Biological Mass Spectrometry, 22, 600-612, 1993.

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Claims

claims

1. A method for determining the ratio of the protein frequencies of similar proteins in a first (a) and at least a second (b) sample, with the steps:

Label the proteins of at least one sample with stable ones

isotopes

Production of a protein mixture (c) of known amounts of protein from the first (a) and the at least second (b) sample, separate separation of the proteins of the first sample (a), the protein mixture (c) and the at least second sample (b) with the aid of a Separation means, in particular a 2D gel,

Determination of the isotopomer distribution of the proteins from samples (a), (b) and (c) by mass spectrometry, in particular by MALDI-TOF mass spectrometry, and calculation of the ratios of the protein frequencies between the first (a) and the at least second (b)

Sample from the determined isotopomer distributions.

2. The method according to claim 1, characterized in that the proteins of the samples are fragmented before or after the separation, in particular by specific cleavage or digestion of the proteins.

3. The method according to claim 1 or 2, characterized in that the samples come from an in vivo system, in particular are an in vivo system.

4. The method according to claim 1, 2 or 3, characterized in that the marking takes place in vivo.

5. The method according to any one of claims 1 to 4, characterized in that at least one sample is influenced, in particular stimulated, with at least one influencing agent before the production of the protein mixture.

6. The method according to claim 5, characterized in that the influencing agent causes a change in the synthesis rate of at least one protein in the sample.

7. The method according to any one of the preceding claims, in particular for determining the rate of incorporation of substances labeled with stable isotopes, in particular amino acids, into proteins with the steps:

Mixing the labeled substances with the cell cultures, obtaining at least one protein harvest by removing the proteins at at least a certain point in time, separating the proteins of the respective protein crops separately with the aid of a separating agent, in particular a 2D gel, optionally obtaining fragments of the separated proteins of the respective Protein crops by specific

Cleavage or digestion, in particular tryptic digestion, of the proteins before or after the separation, determination of the isotopomer distribution of the fragments of the respective protein crops by mass spectrometry, in particular MALDI-TOF mass spectrometry and Calculation of the incorporation rate of the substance from the determined isotopomer distributions of the respective protein crops.

8. Use of the method according to claim 7 for determining the. Effect of at least one influencing, in particular stimulation, on the protein frequency ratios of similar proteins in a first and an at least second labeled sample, at least one of the samples being influenced with at least one influencing agent, characterized in that the rates of incorporation of the substance for the respective sample are determined and the effect of the influencing is determined by forming the ratio of the respective installation rates.

9. A method for determining the accumulation of stable isotopes in molecules, in particular in proteins or peptides, characterized in that the isotopomer distribution of the molecules is determined by mass spectrometry and the accumulation of the stable isotopes by comparison of the ascertained

Isotopomer distribution is calculated with at least one reference distribution of the molecules.

10. The method according to claim 9, characterized in that the reference distribution is the theoretical, in particular naturally occurring isotopomer distribution of the molecules.

11. The method according to claim 10, characterized in that the molecular composition is determined on the basis of the mass fingerprint of the molecules and the theoretical isotopomer distribution of the molecules is determined mathematically on the basis of the molecular composition and / or with the aid of a database.

12. The method according to any one of claims 9 to 11, characterized in that the reference distribution is determined from non-enriched molecules and / or enriched molecules with known enrichment.

13. A method for determining the different enrichment of stable isotopes in proteins, peptides and / or their fragments in different samples, characterized in that the amino acids and / or the fragments of the proteins and / or peptides obtained by hydrolysis and subsequent chromatography, the

Isotope composition of the amino acids and / or the fragments determined and the average of the determined isotope compositions

Enrichment rate is determined.

14. Use or method according to any one of the preceding claims, characterized in that the proteins are labeled by amino acids which have been enriched with stable isotopes.

15. Use or method according to one of the preceding claims, characterized in that the isotopes are ¹⁵ N, ¹⁴ C, ¹³ C, ^1β O, ³⁴ S and / or ² H ₂ .

16. Use or method according to any one of the preceding claims, characterized in that the isotope enrichments, in particular the degree of labeling in the measured labeled peptides and / or proteins, less than 95%, preferably less than 90%, in particular less than 80%, in particular less than 30%.

17. The method according to any one of the preceding claims, characterized in that the ionization ability of molecules to be examined, in particular proteins and / or peptides, by modification of the molecules with a first

Modifying substance is increased.

18. The method according to claim 17, characterized in that the first modification substance is O-methyl-isourea.

19. The method according to claim 17, characterized in that the first modification substance is nicotinyl-N-hydroxysuccinimide.

20. Method for the detection of molecules, in particular phosphoproteins and / or phosphopeptides, the at least one

Contain phospho group, characterized in that the phospho group of the molecules is replaced by sulfur, preferably sulfur-containing alkyl groups, and the detection is carried out by shifting the center of gravity of the isotopomer distribution of the molecules towards heavier masses.

21. The method according to claim 20, characterized in that the ionization ability of the molecules is increased by a second modification substance.

22. The method according to claim 21, characterized in that the second modification substance is an alkylating reagent, preferably 3- (acrylamidopropyl) trimethylammonium chloride.

23. A method for determining qualitative information about proteins and / or peptides in samples, in particular in biological samples with cells and / or cell systems, in particular according to one of the preceding claims, wherein the proteins and / or peptides are labeled with two or more stable isotope-labeled amino acids the process steps:

Treating at least one sample with at least one influencing agent before and / or after the labeling of the proteins and / or peptides in the sample,

Harvesting the cells and / or cell systems, measuring one or more proteins and / or peptides by mass spectrometry, determining the different isotopomer distributions (RIA) for selected or all measured proteins and / or peptides of a sample,

Analysis of the isotopomer distributions to calculate the qualitative information.

24. A method for determining qualitative information about proteins and / or peptides in at least two samples, in particular biological samples with cells and / or cell systems, in particular according to a method according to one of the preceding claims, wherein the proteins and / or peptides with at least one stable isotopically labeled

Amino acid are marked, comprising the method steps: treating each sample with at least one influencing agent before and / or after the labeling of the proteins and / or peptides in the sample, - harvesting the cells and / or cell systems, Measurement of at least two, in particular at least four, preferably at least six, peptides and / or proteins from each sample by mass spectrometry, determination of the different isotopomer distributions (RIA) for selected or all measured proteins and / or peptides in a first and at least a second sample .

25. A method for determining the ratio of the protein frequencies of similar proteins in a first and at least a second sample, in particular a biological sample with cells and / or cell systems, wherein at least one sample is labeled with at least one stable isotope-labeled amino acid, comprising the method steps:

Treating each sample with at least one influencing agent before and / or after the labeling of the proteins and / or peptides in the sample, - harvesting the cells and / or cell systems,

Mix aliquots of samples with the same

Protein content,

Measurement of at least two, in particular at least four, preferably at least six proteins of the individual samples and the mixtures of the samples by

mass spectrometry,

Determination of the different isotopomer distributions for selected or all measured proteins in a first and at least a second sample, - Analysis of the isotopomer distributions to calculate the

Ratio of protein abundances in each sample using the isotopomer distributions of the proteins from the at least two samples and the mixture of the samples.

26. The method according to any one of the preceding claims, characterized in that a mixture of two or more different labeled amino acids, in particular differently labeled amino acids, is used for labeling.

27. The method according to any one of the preceding claims, characterized in that a stable isotope source, which is not an amino acid, is used for labeling, in particular ^d- labeled sugar and / or ¹⁵ N atmosphere.

28. The method according to any one of the preceding claims, characterized in that the peptides and / or proteins according to the

Marking and be fragmented one or more times before the mass spectrometric measurement.

29. The method according to any one of the preceding claims, characterized in that the peptides and / or proteins are separated in one or more separation steps after the treatment with an influencing agent and before the mass spectrometric measurement.

30. The method according to any one of the preceding claims, characterized in that MALDI ionization and / or electrospray ionization for mass spectrometric analysis with two or more quadrupoles, "time of flight", "iontrap" and / or "ioncyclotron" for ion separation is combined.