WO2003098182A2 - Procede de quantification de molecules - Google Patents

Procede de quantification de molecules Download PDF

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Publication number
WO2003098182A2
WO2003098182A2 PCT/EP2003/005091 EP0305091W WO03098182A2 WO 2003098182 A2 WO2003098182 A2 WO 2003098182A2 EP 0305091 W EP0305091 W EP 0305091W WO 03098182 A2 WO03098182 A2 WO 03098182A2
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Prior art keywords
proteins
sample
peptides
protein
isotopomer
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PCT/EP2003/005091
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German (de)
English (en)
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WO2003098182A3 (fr
WO2003098182B1 (fr
Inventor
Werner Stegmann
Michael Cahill
André SCHRATTENHOLZ
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Proteosys Ag
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Priority claimed from DE10315932A external-priority patent/DE10315932A1/de
Application filed by Proteosys Ag filed Critical Proteosys Ag
Priority to AU2003233330A priority Critical patent/AU2003233330A1/en
Publication of WO2003098182A2 publication Critical patent/WO2003098182A2/fr
Publication of WO2003098182A3 publication Critical patent/WO2003098182A3/fr
Publication of WO2003098182B1 publication Critical patent/WO2003098182B1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • 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.
  • the mass spectrometric methods used are MALDI-TOF mass spectrometry and ESI mass spectrometry.
  • MALDI matrix-assisted laser desorption ionization
  • ESI electro-spray ionization
  • Mass analyzers are used, which can examine ions according to their mass to charge ratio m / z.
  • 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.
  • leucine (Leu-d10) labeled with stable isotopes is incorporated into bacteria.
  • leucine (Leu-d3) labeled with stable isotopes is incorporated into mammalian cell cultures.
  • Mass spectrometry can be obtained.
  • 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.
  • an accurate quantification of the labeled and unlabeled cells is not possible without a complete incorporation of Leu-d3 in the proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reference distribution is determined from non-enriched molecules and / or enriched molecules with known enrichment.
  • 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.
  • 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.
  • the method for determining the accumulation of stable isotopes in proteins in a sample comprises the following steps:
  • 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,
  • 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.
  • the ICP Inductively Coupied Plasma
  • MS is suitable for determining the isotope compositions.
  • 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.
  • 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.
  • the isotopomer distributions come about through the existence of heavy isotopes of the elements from which the molecules are built.
  • 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.
  • MALDI-TOF mass spectrometry enables the isotopomer distributions of peptides to be determined reproducibly within narrow error limits.
  • 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:
  • 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,
  • 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.
  • 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.
  • 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, 1 ⁇ O, M S and / or 2 H 2 . Labeling "in vitro" is also possible, for example by protein alkylation.
  • a protein mixture of known amounts of protein from the first and the at least second sample manufactured 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Isotopomer distributions of the fragments can be determined in high resolution, reproducibly within narrow error limits. This is a basic requirement of the procedure.
  • 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.
  • 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 monoisotopic peaks of the protein or peptide molecules are the peaks with the lowest m / z values in an isotopomer distribution.
  • RIA values Relative Isotopologue Abundance values
  • RIA values Relative Isotopologue Abundance values
  • an RIA value of an isotopologist Mi of a specific protein or peptide fragment p results from:
  • the ion signals are determined from integrals via the corresponding peaks.
  • the mole fraction C is determined according to [Vogt 1993]:
  • 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.
  • 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
  • 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:
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a separating agent in particular a 2D gel.
  • 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.
  • 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.
  • 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.
  • the proteins are labeled by amino acids which have been enriched with stable isotopes.
  • 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.
  • the isotopes are 15 N, 14 C,
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the lysine peptides are severely disadvantaged compared to the arginine peptides.
  • a first modification substance 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.
  • O-methylisourea or nicotinyl-N-hydroxysuccinimide [James 2000] are used for both C- and N-terminal modifications.
  • the molecules to be examined 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.
  • the introduced sulfur-containing group in particular a sulfhydryl group
  • 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
  • a basic group for example, a phosphate group
  • Michael addition takes place before, for example, tryptic digestion
  • new t ⁇ / ptic cleavage sites can be introduced.
  • 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.
  • 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.
  • synthetic or artificially synthesized polypeptides or proteins can also be produced as long as they are also synthesized by ribosomes.
  • 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.
  • 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.
  • the invention covers methods in which individual protein abundances of non-synthetically produced polypeptides or proteins are determined in at least one sample.
  • 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.
  • 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.
  • the different samples in particular the different cell cultures or animals or groups of animals, are treated differently during the course of the experiment.
  • the experimental conditions should be chosen accordingly.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • this aspect of the invention also relates to the method according to the invention with regard to qualitative 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.
  • two or more proteins or peptides in one or more samples.
  • even more than 10 or even more than 100 different proteins can be analyzed in one or more samples according to 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.
  • Fig. 1 Measured with MALDI-TOF mass spectrometry
  • 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
  • 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.
  • a) is a published method: Oda et al., 1999; Pasa-Tolic et al., 1999; Smith et al., 2001.
  • 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.
  • 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.
  • Cell pool A labeled cells
  • Cell pool B unlabeled cells a) The cells from cell pool A are grown in a medium which
  • the 15 N-enriched amino acids are taken up by the cells and into the
  • Cell pool B 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
  • 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.
  • Cell pool A labeled cells
  • Cell pool B unlabeled cells
  • Cell pool C unlabeled cells
  • 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.
  • mixture AB mixture of equal amounts of protein from cell pool A and cell pool B.
  • 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.
  • 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.
  • 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 ⁇ , Bß, B t3 , ...
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • the mouse 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.
  • 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.
  • 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.
  • Proboscis DH and Edmondson RD High-resolution mass spectrometry and accurate mass measurements with emphasis on the characterization of peptides and proteins by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. J. Mass Spectrometry, 32, 263-276, 1997.

Abstract

La présente invention concerne un procédé de détermination du rapport des fréquences protéiques de protéines du même type dans un premier et au moins un deuxième échantillon, un procédé de détermination de l'enrichissement d'isotopes stables dans des molécules, et un procédé de détermination du taux d'intégration de substances marquées dans des protéines.
PCT/EP2003/005091 2002-05-15 2003-05-15 Procede de quantification de molecules WO2003098182A2 (fr)

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GB2413695A (en) * 2004-04-30 2005-11-02 Micromass Ltd Mass spectrometer
EP1635178A2 (fr) * 2004-09-14 2006-03-15 ProteoSys AG Protéine du cancer du sein
CN1314968C (zh) * 2004-03-11 2007-05-09 复旦大学 痕量多肽或蛋白质富集及其直接分析方法
US8515685B2 (en) 2004-04-30 2013-08-20 Micromass Uk Limited Method of mass spectrometry, a mass spectrometer, and probabilistic method of clustering data
CN112683986A (zh) * 2021-03-18 2021-04-20 裕菁科技(上海)有限公司 一种用于定量样品中目标分析物的天然同位素校准曲线法

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005059538A1 (fr) * 2003-12-17 2005-06-30 Yoshio Yamauchi Procede d'analyse de proteines
CN1314968C (zh) * 2004-03-11 2007-05-09 复旦大学 痕量多肽或蛋白质富集及其直接分析方法
GB2413695A (en) * 2004-04-30 2005-11-02 Micromass Ltd Mass spectrometer
GB2413695B (en) * 2004-04-30 2009-01-21 Micromass Ltd Mass spectrometer
US8012764B2 (en) 2004-04-30 2011-09-06 Micromass Uk Limited Mass spectrometer
US8515685B2 (en) 2004-04-30 2013-08-20 Micromass Uk Limited Method of mass spectrometry, a mass spectrometer, and probabilistic method of clustering data
EP1635178A2 (fr) * 2004-09-14 2006-03-15 ProteoSys AG Protéine du cancer du sein
WO2006029836A2 (fr) * 2004-09-14 2006-03-23 Proteosys Ag Proteines de cancer du sein
EP1635178A3 (fr) * 2004-09-14 2006-05-24 ProteoSys AG Proteine du cancer du sein
WO2006029836A3 (fr) * 2004-09-14 2006-07-20 Proteosys Ag Proteines de cancer du sein
CN112683986A (zh) * 2021-03-18 2021-04-20 裕菁科技(上海)有限公司 一种用于定量样品中目标分析物的天然同位素校准曲线法
CN112683986B (zh) * 2021-03-18 2021-06-15 裕菁科技(上海)有限公司 一种用于定量样品中目标分析物的天然同位素校准曲线法

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