WO2012111249A1 - Procédé de détection d'un changement de masse dans un procédé de spectrométrie de masse et procédé de quantification de la quantité absolue d'une protéine stable marquée par un isotope - Google Patents

Procédé de détection d'un changement de masse dans un procédé de spectrométrie de masse et procédé de quantification de la quantité absolue d'une protéine stable marquée par un isotope Download PDF

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WO2012111249A1
WO2012111249A1 PCT/JP2012/000227 JP2012000227W WO2012111249A1 WO 2012111249 A1 WO2012111249 A1 WO 2012111249A1 JP 2012000227 W JP2012000227 W JP 2012000227W WO 2012111249 A1 WO2012111249 A1 WO 2012111249A1
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protein
labeled
stable isotope
target protein
tag
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PCT/JP2012/000227
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Japanese (ja)
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潤一 上家
裕貴 川上
元栄 坂上
欣二 代田
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学校法人麻布獣医学園
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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

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  • the present invention relates to a method of using a stable isotope-labeled protein as an internal standard in an analysis using a mass spectrometer, and more specifically, by analyzing a plurality of peptide fragments derived from a target protein at a time in a biological sample. Quantitatively detect unknown post-translational modifications by detecting mass changes in mass spectrometry of target proteins in a sample by quantifying multiple peptide fragments from a target protein at once, with a highly sensitive detection method
  • the present invention also relates to a method for identifying and / or quantifying and / or identifying a sequence abnormality of a target protein due to a gene mutation.
  • the human genome project has comprehensively analyzed human gene sequences and identified genes that cause diseases one after another.
  • many unexplained diseases remain, and detailed analysis of protein post-translational modifications and single nucleotide polymorphisms (SNPs) is expected in the future.
  • post-translational modification of proteins that are considered to determine differences in race and individuality, and to determine the constitution of individuals such as disease and drug resistance.
  • Analysis of single nucleotide polymorphisms (SNPs) and the like has become more important, and a method for identifying and / or quantifying these has been required.
  • antibodies that detect post-translational modifications there are antibodies that can detect phosphorylation of tylosin, serine, and threonine, but examples where detection sensitivity is not sufficient, examples in which phosphorylation of each of these amino acid residues cannot be distinguished, Depending on the three-dimensional structure of the protein, there are problems such as the fact that these phosphorylations cannot always be detected. In addition, it is more difficult to produce an antibody that detects phosphorylation of a specific protein on a specific amino acid residue. Furthermore, detection of proteins and post-translational modifications using antibodies often poses a major problem in that antibodies often react with proteins other than the target protein.
  • the non-specificity of this antibody is one of the major causes that hinders the increase in the accuracy of protein quantification in protein quantification using antibodies such as ELISA. Therefore, instead of the method using these antibodies, a technique for quantifying proteins and post-translational modifications with high accuracy using mass spectrometry is being developed.
  • Mass spectrometry is a method in which a sample is ionized and the ionized molecules are separated and detected according to mass / charge (m / z). This method is used for detection and measurement of various biological materials. Have been used. Recent advances in mass spectrometry have enabled the use of various ionization methods such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), and ion traps. Various mass spectrometers have been developed using various analyzers that analyze ionized samples by the time of flight method (TOF: Timeof ⁇ Flight), quadrupole method, Fourier transform method, and the like.
  • TOF Timeof ⁇ Flight
  • mass spectrometers with various functions such as liquid chromatography mass spectrometer (LC-MS) connected with liquid chromatography and tandem mass spectrometer (MS / MS spectrum) with two mass spectrometers combined. A combination of these functions is used for detection, measurement, and quantification of biological materials (Patent Documents 1 to 3).
  • LC-MS liquid chromatography mass spectrometer
  • MS / MS spectrum tandem mass spectrometer
  • a conventional protein quantification method using mass spectrometry is a method of selecting a peptide fragment to be quantified in a target protein and quantifying the amount of the peptide fragment. That is, as shown in FIG. 1, (1) a step of selecting an arbitrary peptide fragment to be quantified in the target protein; (2) each peptide fragment at a predetermined concentration stage and an amino acid sequence corresponding to a specific amount of the peptide fragment A step of carrying out mass spectrometry for a mixture of a certain stable isotope-labeled internal standard peptide fragment and calculating a mass spectrum area ratio of unlabeled peptide fragment / stable isotope-labeled internal standard peptide fragment, respectively, and preparing a calibration curve; 3) Mass spectrometry is performed on the sample containing the target protein subjected to the fragmentation treatment and the sample containing the specific amount of stable isotope-labeled peptide fragment, and the unlabeled peptide fragment / stable isotope-labele
  • the conventional protein quantification method using mass spectrometry has a problem that the peptide fragment to be quantified must be selected.
  • the ease of ionization of peptide fragments varies from peptide fragment to peptide fragment, and in order to quantify proteins by mass spectrometry, it is very important to determine which sequence of peptide fragments to target, and for various selections. Although criteria are being studied, the selection of peptide fragments to be quantified is a difficult process at present. Further, in the conventional method, after fragmenting the target protein in the sample, a stable isotope-labeled internal standard peptide fragment is added and subjected to mass spectrometry.
  • JP 2004-28993 A Japanese Patent Laid-Open No. 2004-77276 JP-T-2004-533610 WO00 / 67017 JP 2010-210461 A
  • An object of the present invention is to identify and / or quantify a post-translational modification of a protein using a mass spectrometer, to identify and / or quantify a genetic mutation of a protein, and to detect a protein with high sensitivity and high accuracy therefor. It is to provide a quantitative method.
  • the present invention provides [1] (a) peptide fragmentation in a predetermined concentration step or a mixture of each target protein in a sample or a target protein in a sample and a stable isotope labeled protein corresponding to a specific amount of the target protein.
  • a method for detecting a mass change in mass spectrometry of a target protein in a sample comprising a) to (c), and [2] detection of a mass change in mass spectrometry of a target protein is a post-translational modification of the target protein.
  • the method according to [1] above which is the identification of a site abnormality in a target protein due to a gene mutation, and [7] the sequence abnormality in the target protein due to a gene mutation is caused by single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • the method according to [5] or [6] above, or [8] a stable isotope-labeled protein having no post-translational modification or gene mutation as the stable isotope-labeled protein The present invention relates to the method according to any one of [1] to [7].
  • the post-translational modification site or gene mutation site is (1) a site where amino acid sequence changes due to post-translational modification or gene mutation are published in public databases; (2) serine or It is a sequence site containing threonine and tylosin; (3) It is a sequence site containing asparagine at the extracellular position of a membrane protein or secreted protein; (4) It is a sequence site containing lysine; (5) Cysteine (6) a sequence site containing glutamic acid; (7) a sequence site containing proline; and (8) a quantitative value determined by the method described in [1] above is statistical.
  • the present invention further provides [15] (i) a step of synthesizing a stable isotope-labeled tag protein fusion protein using a stable isotope-labeled amino acid; (ii) the stable isotope-labeled tag protein fusion protein, and a specific amount of Peptide fragmentation treatment is performed on the mixture with the non-stable isotope-labeled tag protein, mass analysis is performed on the resulting peptide fragment group, and the peptide fragment derived from the tag protein portion of the stable isotope-labeled tag protein fusion protein / unstable Quantifying each peptide fragment derived from the tag from the signal area ratio or signal intensity ratio of the peptide fragment derived from the isotope-labeled tag protein; (iii) all the peptide fragments derived from the tag protein of the stable isotope-labeled tag protein fusion protein The average value of the quantitative values of the synthesized stable isotope-labeled tag protein fusion tag A method
  • (IV) a sequence containing lysine; (V) any protein digestion enzyme selected from trypsin, glutamyl peptidase, asparagine peptidase, and chymotrypsin. (VI) a sequence that is fragmented into a peptide using a chemical substance; an amino acid sequence that is set based on any of the conditions (I) to (VI)
  • the quantitative method according to any one of the above [15] to [17], or [19] the amount of the non-stable isotope-labeled tag protein is determined by amino acid analysis [15]
  • the amount of the non-stable isotope-labeled tag protein is determined by a biochemical colorimetric method. ] To the quantification method according to any one of [19].
  • the protein since it is not necessary to select the target peptide fragment, the protein can be quantified more easily and more sensitively and more accurately than the conventional method in order to quantify a plurality of peptide fragments at once. Can do.
  • the protein can be quantified at a cost of about 1/20 that was required for the measurement using a conventional peptide fragment.
  • proteins can be quantified with higher accuracy than ELISA methods using conventional antibodies. Identification and translation of unknown post-translational modification sites of proteins and single nucleotide polymorphisms that have been difficult in the past The post-modification rate can be quantified.
  • the present invention can identify and quantify post-translational modifications and identify genetic mutations simultaneously with quantitative analysis of proteins, the analysis time per protein can be dramatically reduced, and multiple molecules such as screening can be performed simultaneously. It also has the effect that it can be used effectively for analysis. That is, the method of the present invention, which is particularly excellent in quantitative accuracy, cost, post-translational modification quantification, and simplicity, can be said to be a technology that contributes to the fields of life science and medical technology in place of conventional methods (Table 1).
  • the quantitative value is the peptide fragment not subjected to post-translational modification, and the amount obtained by subtracting the quantitative value from the average quantitative value is the peptide subjected to post-translational modification. Calculated as the amount of fragments.
  • mass change in mass spectrometry of target protein means that the mass of the target protein and / or peptide fragment derived from the target protein is a database or information obtained by actually confirming the DNA sequence or amino acid sequence. This means that it differs from the mass calculated based on it, and does not necessarily mean that a substantial mass change is detected.
  • detect means quantification and / or site identification, and the screening method for the “mass change in mass spectrometry of target protein” region also includes mass change in mass spectrometry in the target protein. Is included in the “detection” of the present invention.
  • detection of post-translational modification means “quantification of post-translational modification” and “identification of post-translational modification site”
  • detection of gene mutation means “quantification of gene mutation” and “identification of gene mutation site”.
  • Examples of the “mass change in target protein mass spectrometry” in the present invention include post-translational modification of the target protein, target protein sequence abnormality due to gene mutation, splicing variant, and protein cleavage or degradation by protease.
  • a target protein sequence abnormality due to post-translational modification or gene mutation of the target protein can be preferably exemplified.
  • the post-translational modification is not particularly limited as long as it is a post-translational modification of the protein.
  • Glycation (glycosylation), phosphorylation, methylation, acylation, alkylation, dimethylation, biotinylation, formylation, carboxyl , Glutamylation, glycylation, hydroxylation, iodination, isoprenylation, lipoylation, prenylation, GPI anchor formation, ADP ribosylation, FAD coupling, polyethylene glycolation, phosphatidylinositol addition, phosphopantetheinylation, pyroglutamic acid Formation, racemization, tylosin sulfate, selenoylation, ISGization, SUMOylation, ubiquitination, NEDDization and the like.
  • target protein sequence abnormalities due to gene mutations include single nucleotide polymorphisms (SNPs), gene sequence duplication, deletion, insertion, differences in the number of repeated sequences, the number of transposons such as LINE and SINE, etc. Examples include differences caused by gene sequence mutations such as differences and transposon insertions and deletions.
  • SNPs single nucleotide polymorphisms
  • single nucleotide polymorphisms can be preferably exemplified.
  • the mass change site such as the post-translational modification site or the gene mutation site is set according to any one of the following conditions (1) to (8).
  • a site where changes in amino acid sequence due to post-translational modification or gene mutation are publicly disclosed in public databases; (2) a sequence site containing serine, threonine or tylosin; (3) an extracellular site of a membrane protein or a sequence site containing asparagine in a secreted protein; (4) a sequence site containing lysine; (5) a sequence site containing cysteine; (6) a sequence site containing glutamic acid; (7) a sequence site containing proline; (8)
  • the quantitative value obtained by the method of the present invention is a sequence site that shows an outlier from the average quantitative value by a statistical method;
  • the “predetermined concentration step or each target protein in a sample” of the present invention may be a target protein having a known concentration or a target protein in a sample to be measured, and the concentration of the target protein in the sample is often unknown.
  • target proteins with known concentrations include those produced by cell-free systems such as cells, Escherichia coli, and wheat germ by genetic engineering techniques, purified and extracted, and measured for concentrations, and commercially available products.
  • the sample can be exemplified by a biological sample such as a cell extract, a tissue extract, a culture solution, or a body fluid such as blood or spinal fluid.
  • the “predetermined concentration step” can be appropriately set according to the amount of target protein in the sample to be measured, the purification method, and the sensitivity and accuracy of the mass spectrometer, for example, 10 fmol, 50 fmol, 100 fmol, 500 fmol, 1000 fmol concentration steps. It can also be.
  • the “stable isotope labeled protein corresponding to a specific amount of the target protein” of the present invention (hereinafter, also simply referred to as “stable isotope labeled protein” or “stable isotope labeled internal standard protein”) It is a protein having the same amino acid sequence, and may be one that is labeled with a stable isotope by including one or more kinds of stable isotope elements.
  • the nitrogen stable isotope 15 N examples thereof include a carbon stable isotope 13 C, an oxygen stable isotope 18 O, and a hydrogen stable isotope 2 H.
  • a stable isotope-labeled protein can be artificially chemically synthesized using an amino acid containing a stable isotope-labeled element, or cells or E. coli are cultured in a culture solution containing a stable isotope-labeled element.
  • a stable isotope-labeled protein can be produced in a cell-free system, and such stable isotope-labeled protein may not have post-translational modifications or gene mutations. preferable.
  • the “specific amount” is not particularly limited, and can be any amount depending on the amount of target protein in the sample to be measured, the purification method, the sensitivity and accuracy of the mass spectrometer, for example, 500 fmol. Can do.
  • the concentration of the stable isotope labeled protein can also be measured using the method for quantifying the stable isotope labeled protein of the present invention.
  • the “peptide fragmentation treatment” of the present invention can be used as long as it can cleave proteins to produce peptide fragments. Fragmentation treatment using chemical substances and fragmentation treatment methods using protein digestive enzymes Can be mentioned.
  • the method for detecting a mass change in mass spectrometry of a target protein in a sample of the present invention (hereinafter also referred to as “method of the present invention”) is to perform fragmentation treatment of the target protein and the internal standard protein in the same solution.
  • the target protein and the internal standard protein are subjected to fragmentation treatment under the same conditions. Therefore, a fragmentation method using a chemical substance, which is generally said to be inferior in reproducibility compared with protein digestion enzymes, can also be used.
  • Chemical substances used for fragmentation include acidic compounds such as hydrochloric acid, sulfuric acid, trifluoroacetic acid, citric acid, malic acid and aspartic acid, alkaline compounds such as sodium hydroxide and potassium hydroxide, and cyanogen bromide. Can be mentioned.
  • protein digestion enzymes used for fragmentation include endopeptidases and exopeptidases, and more specifically serine proteases such as chymotrypsin and subtilisin, pepsin, cathepsin D, and HIV protease.
  • Examples include aspartic protease, metalloprotease such as thermolysin, cysteine protease such as papain and caspase, N-terminal threonine protease, glutamate protease, arginine endopeptidase, among others, trypsin, Preferable examples include glutamyl peptidase, asparagine peptidase and chymotrypsin. These peptide fragmentation methods can also be performed by combining two or more methods.
  • a region included in a peptide fragment that could not be detected by a certain peptide fragmentation method is also subjected to mass spectrometry as a different peptide fragment depending on another peptide fragmentation method or a combination of a plurality of fragmentation methods. , May be detectable. Therefore, by using a plurality of peptide fragmentation methods, it is possible to improve the area detected in the target protein mass spectrometry, that is, the detection coverage.
  • the mass spectrometry used in the present invention is not particularly limited as long as it is a method of ionizing a sample and separating and detecting ionized molecules according to mass / charge (m / z). Electrospray ionization (ESI) ) And matrix-assisted laser desorption / ionization (MALDI), ionization by ion trap, time-of-flight (TOF), quadrupole, Fourier transform, etc. A method for analyzing a sample can be mentioned.
  • the mass spectrometer may be used alone, or may be connected to a separation instrument such as liquid chromatography or a measuring instrument, and two liquid chromatography mass spectrometers (LC-MS) and mass spectrometers are used.
  • a tandem mass spectrometer MS / MS spectrum
  • LC-MS / MS liquid chromatography tandem mass spectrometer
  • LC-MS / MS can be preferably exemplified.
  • one or more other separation devices, measurement devices, and the like may be appropriately disposed between a device such as liquid chromatography and a mass spectrometer or tandem mass spectrometer.
  • the target protein is quantified.
  • the peptide fragment quantification here is the signal of the target protein-derived peptide fragment / stable isotope-labeled protein-derived peptide fragment, even if it is an absolute amount obtained by creating a calibration curve described later and calculating the quantitative value. It may be a relative amount obtained from an area ratio or a signal intensity ratio.
  • measurement is performed by mass spectrometry using each target protein (also referred to as “artificial standard protein”) at a predetermined concentration step with known concentration and a stable isotope-labeled internal standard protein.
  • a method of preparing a calibration curve and calculating the absolute amount of the target protein in the sample as a quantitative value can be mentioned (FIG. 2).
  • the calibration curve may be prepared in advance.
  • a method for measuring the amount of a target protein in a sample using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS) comprising the following steps (A) to (C) is preferably exemplified. Can do.
  • step (A) Peptide fragmentation treatment is performed on a mixture of each target protein at a predetermined concentration step and a stable isotope-labeled protein corresponding to a specific amount of the target protein, and LC-MS / Performing mass spectrometry using MS, and calculating a signal area ratio and a signal intensity ratio of the target protein-derived peptide fragment / stable isotope-labeled protein-derived peptide fragment, respectively, and creating a calibration curve;
  • step (B) After adding the specific amount of stable isotope-labeled protein to the sample containing the target protein, the peptide fragmentation treatment in step (A) was performed, and LC-MS / MS was used for the obtained peptide fragment group Conducting mass spectrometry and calculating the mass spectrum area ratio and the intensity ratio of the target protein-derived peptide fragment / stable isotope-labeled protein-derived peptide fragment, respectively;
  • C) A step of quantifying each target protein-derived peptide fragment in
  • all the peptide fragments derived from the target protein are quantified by the protein quantification method using the mass spectrometer, and all the target protein-derived peptides are quantified.
  • the amount of the peptide fragment in which mass change has occurred in mass spectrometry is used. I just need it.
  • the method includes the following steps (a) to (c) and quantifies a change in mass in a mass spectrometry of a target protein in a sample.
  • A) Peptide fragmentation treatment is performed on a mixture of each target protein in a predetermined concentration step or each sample and a stable amount of a stable isotope-labeled protein corresponding to the target protein.
  • a protein quantification method using the mass spectrometer and / or a method for quantifying a mass change in the mass spectrometry method of the present invention The average value of the quantitative values of all the target protein-derived peptide fragments is obtained as an average quantitative value, and the average quantitative value is compared with the quantitative values of all the target protein-derived peptide fragments, and an outlier is obtained by a statistical method. Any method may be used as long as the target protein-derived peptide fragment to be shown is selected. Or you may select the target protein origin peptide fragment which shows an outlier by a statistical method based on the ratio of the mass change in mass spectrometry.
  • Such a statistical method is not particularly limited, and examples thereof include a Suminorf Grubbs test.
  • identifying a peptide fragment that shows a mass change in mass spectrometry in the target protein it is possible to screen for a mass change in mass spectrometry of the target protein.
  • a method for identifying a mass change in mass spectrometry of a target protein in a sample comprising the following steps (a ′) to (c ′) can be exemplified.
  • a ′ Peptide fragmentation treatment is performed on a mixture of each target protein in a predetermined concentration step or sample and a stable amount of a stable isotope-labeled protein corresponding to the target protein, and a peptide fragment group obtained is obtained.
  • the method for detecting mass change in the mass spectrometry method of the present invention compares the analysis result of the mass change in the mass spectrometry method of the protein in the sample of the healthy subject and the disease patient, thereby determining the gene causing the disease or the disease. It can be used to identify related genes. Further, by detecting a mass change in mass spectrometry using a disease causal gene or the like as a target protein, it can be used for elucidation of a disease mechanism or prediction of drug efficacy.
  • human EGFR (Epidermal®Growth®Factor®Receptor) has been identified as one of the causative genes of lung cancer, and is a target molecule of the lung cancer therapeutic drug gefitinib (trade name Iressa (registered trademark), manufactured by AstraZeneca).
  • the lung cancer drug gefitinib has an excellent therapeutic effect on one group of patients, but it is known to cause severe side effects in other groups of patients and has become a social problem. This difference in the efficacy of gefitinib is considered to be different due to post-translational modifications such as EGFR gene mutation and EGFR protein phosphorylation in the patient.
  • human EGFR can quantify and / or identify changes in amino acid sequences due to post-translational modifications to each amino acid shown in Table 2 and gene mutations shown in Table 3.
  • the method for quantifying the absolute amount of the stable isotope-labeled protein of the present invention is used in the method for detecting the absolute amount of the stable isotope-labeled protein used as an internal standard in mass spectrometry and the mass change in the mass spectrometry of the present invention. It can be used to measure the absolute amount of stable isotope labeled protein.
  • a step of synthesizing a stable isotope-labeled tag protein fusion protein using a stable isotope amino acid (I) a step of synthesizing a stable isotope-labeled tag protein fusion protein using a stable isotope amino acid; (Ii) Peptide fragmentation treatment is applied to a mixture of the stable isotope-labeled tag protein fusion protein and a specific amount of the non-stable isotope-labeled tag protein, and the resulting peptide fragment group is subjected to mass spectrometry and stable.
  • the method is not particularly limited as long as it is a method for quantifying a stable isotope-labeled protein used as an internal standard in mass spectrometry, comprising the steps (i) to (iii).
  • the stable isotope-labeled tag protein at a predetermined concentration step is used in the same manner as in the steps (A) to (C) of the method for measuring the amount of the target protein in the sample.
  • the absolute amount can be calculated using a calibration curve prepared by mass spectrometry using a specific amount of non-stable isotope-labeled tag protein. Such a calibration curve may be prepared in advance.
  • a stable isotope labeled tag protein fusion protein can be quantified using an unstable stable isotope labeled tag protein of known concentration as an internal standard.
  • the tag protein of the stable isotope-labeled tag protein and the stable isotope-labeled tag protein fusion protein may be of the same type, and the tag protein of the stable isotope-labeled tag protein fusion protein. Any protein can be used as the protein to be fused.
  • the stable isotope-labeled tag protein fusion protein may be a protein having a structure in which a tag protein of the same type as the non-stable isotope-labeled tag protein is fused to the N-terminus or C-terminus of any protein.
  • a stable isotope-labeled tag protein fusion protein can be produced using genetic engineering or chemical synthesis technology.
  • a tag in which the DNA sequence of the tag protein sequence is linked to the N-terminus or C-terminus of the DNA sequence of any protein It can also be synthesized by expressing a protein fusion protein expression vector in the presence of a stable isotope-labeled amino acid.
  • Such amount can be quantified by a biochemical colorimetric method such as amino acid analysis method, Bradford method, Biuret method, Lowry method, BCA (bicinchoninic acid) method or the like.
  • the tag protein may be any protein that can obtain a peptide fragment detectable by mass spectrometry by enzymatic digestion or chemical fragmentation.
  • glutathione-S-transferase GST: Glutathione-S-transferase
  • Histidine His
  • MBP maltose binding protein
  • c-Myc HA tag
  • FLAG tag FLAG tag
  • GFP tag etc.
  • GST tag SEQ ID NO: 7
  • His tag SEQ ID NO: 8
  • the His tag In addition to the GST tag consisting of the amino acid sequence of SEQ ID NO: 7, amino acid sequences having substitutions, insertions and deletions in such a sequence, and those consisting of GST amino acid sequences published in various databases such as NCBI can also be used.
  • the His tag a His tag having an arbitrary length in which 3 to 15 histidines are linked can also be used.
  • the tag protein may be a single type of tag protein, or may be a plurality of the same tag protein or different types of tag proteins, or may be repeatedly linked.
  • a stable isotope-labeled tag protein fusion protein obtained by fusing a stable isotope-labeled protein to the N-terminal side or C-terminal side of the stable isotope-labeled protein.
  • stable isotope-labeled tag protein Using the tag protein not labeled with a stable isotope as an internal standard, analyzing the peptide fragment derived from the tag protein portion of the stable isotope-labeled tag protein fusion protein by mass spectrometry, stable isotope-labeled tag protein The absolute amount of the tag protein of the fusion protein can be measured, and the quantitative value of the tag protein can be used as the quantitative value of the stable isotope-labeled protein.
  • a stable isotope-labeled tag protein fusion protein can be used as a stable isotope-labeled protein in the method for detecting mass change in the mass spectrometry of the target protein in the sample of the present invention.
  • Nephrin artificial standard protein was prepared by the following method. Escherichia coli (BL21-CodonPlus (DE3) -RIPL) transformed with pET-vector inserted with Nephrin (swissprot accession No.Q9R044) cDNA was added to kanamycin and chloramphenicol at 30 mg / L and 50 mg / L, respectively. The LB medium was shaken overnight and then diluted in LB medium. D. Culturing was continued until 600 nm showed a value of around 0.4, IPTG was added at a final concentration of 100 mM, and further cultured for 3 hours to induce protein expression.
  • the induced Escherichia coli was subjected to ultrasonic disruption in the presence of 8M urea to prepare a soluble fraction and an insoluble fraction, then fractionated by SDS-PAGE, and detected by CBB R-250.
  • the Escherichia coli soluble fraction was added to a spin column packed with cobalt resin, and the column was washed with a 10 mM imidazole solution, and then the artificial standard protein was eluted with 50 mM, 150 mM, and 500 mM imidazole solutions.
  • Nephrin's stable isotope-labeled protein was prepared by the following method. Escherichia coli (Rosseta (DE3), manufactured by Novagen) transformed with pET-vector with the full-length amino acid sequence of Nephrin (swissprot accession No.Q9R044) with GST and His tag added, magnesium sulfate heptahydrate added (Final concentration: 0.25 g / L) H. L. medium (manufactured by Chlorella Kogyo) at 37 ° C. D. Culturing was performed until 600 nm reached 0.7.
  • LC-MS / MS measurement was performed using 1/5 volume of the peptide sample.
  • GLVQPTR, LTQSMAIIR, DFETLK, VDFLSK , LPEMLK, for IEAIPQIDK stable isotopes (15 N) labeled peptide and a non-labeled peptide was measured with SRM mode LC-MS / MS .
  • concentration of the stable isotope labeled GST in the sample from the peak area ratio of the stable isotope labeled and unlabeled it was 103.5 ⁇ 4.93 fmol / assay.
  • Table 5 shows the results of detection of peptide fragments of stable isotope labeled and unlabeled GST (swissprot accession No. P08515) in the stable isotope labeled GST and His tag fusion Nephrin protein samples.
  • the average value of the ratio of labeled to unlabeled in the 6 peptide fragments measured was 0.36 ⁇ 0.02, and the CV value (%) indicating the reliability of the measured value was 4.4%. That is, it was shown that GST protein can be quantified with high accuracy by using mass spectrometry.
  • stable isotope-labeled GST protein is present in an equimolar amount with the fusion protein in the expressed protein sample, by quantifying stable isotope-labeled GST protein, all stable isotope-labeled GST fusion proteins can be analyzed using mass spectrometry. Quantification is possible.
  • Nephrin protein was quantified by the following method using a stable isotope-labeled internal standard protein. 500 fmol of stable isotope-labeled internal standard protein was added to 10 fmol, 50 fmol, 100 fmol, 500 fmol, and 1000 fmol of artificial standard protein, respectively, to prepare a calibration curve sample. In addition, a measurement sample was prepared by adding 500 fmol of a stable isotope-labeled internal standard protein to a rat glomerular sample.
  • a conventional protein quantification method using a mass spectrometer is a method for quantifying a protein by detecting a specific peptide fragment of a target protein (FIG. 1). Therefore, in the conventional method, Nephrin protein was quantified using GGNPPATLQWLK (SEQ ID NO: 4), which is a partial sequence of Nephrin, as a target peptide fragment. Nephrin artificial standard peptide was prepared by the following method. A partial amino acid sequence GGNPPATLQWLK (SEQ ID NO: 4) of Nephrin (swissprot accession No.
  • Q9R044) was chemically synthesized using a solid phase method, and the peptide concentration in the sample was quantified by amino acid analysis.
  • a peptide GGNPPATLQWL * K (stable isotope labeled L *) containing the stable isotope-labeled leucine was chemically synthesized using the solid phase method, and the peptide concentration was quantified by amino acid analysis.
  • Stable isotope-labeled internal standard peptide fragment GGNPPATLQWL * K (L * is stable isotope) consisting of the same amino acid sequence as the artificial standard peptide fragment consisting of the amino acid sequence of GGNPPATLQWLK (SEQ ID NO: 4) Label) was used to quantify the Nephrin protein in the sample.
  • Samples for preparing a calibration curve were prepared by adding 500 fmol of stable isotope-labeled peptide fragments to 10 fmol, 50 fmol, 100 fmol, 500 fmol and 1000 fmol artificial standard (unlabeled) peptide fragments, respectively.
  • rat glomerular samples were denatured with 7 M guanidine hydrochloride solution (dissolved in 0.1 M Tris-HCl, 10 mM EDTA pH 8.5) to reduce the SH group of the cysteine residue and to reduce iodoacetamide with DTT.
  • Carbamide methylation treatment was carried out. Subsequently, it was desalted and concentrated by methanol chloroform precipitation, and resuspended in 1.2 M urea / 10 mM Tris-HCl. Thereafter, trypsin in an amount of 1/100 of the protein weight was added and fragmented by enzymatic digestion at 37 degrees for 16 hours.
  • the quantitative value of Nephrin protein in the sample was quantified as 66.4 ⁇ 6.1 fmol / assay in the method of the present invention and 51.0 ⁇ 5.6 fmol / assay in the conventional method, It was only quantified as about 76% protein compared to the inventive method (FIG. 4).
  • the conventional method the undigested rate of the sample protein during the enzyme digestion process and the sample loss due to adsorption of the target protein to the tube are not corrected by the stable isotope-labeled internal standard peptide fragment. As a result, the conventional method is underestimated. It is considered that the amount of protein was calculated as a quantitative value.
  • the method of the present invention shows a higher quantitative value than the conventional method.
  • FIG. 5 shows a graph in which the quantitative value of each detected peptide fragment is plotted on the vertical axis with respect to the amino acid sequence number of the Nephrin protein on the horizontal axis. Average quantitative values are indicated by dotted lines. Since peptide fragments having a mass different from that of the amino acid sequence of Nephrin protein are not detected, peptide fragments whose mass is changed by post-translational modification or gene mutation are not detected. Therefore, when the quantitative value of the peptide fragment is statistically significantly smaller than the average quantitative value, the quantitative value of the peptide fragment is calculated from the amount of peptide fragment that has not undergone post-translational modification, and the quantitative value of the peptide fragment from the average quantitative value.
  • the subtracted amount can be calculated as the amount of peptide fragment that has undergone post-translational modification (FIG. 5).
  • the outliers of each peptide fragment relative to the average quantitative value were determined by the Suminorf Grubbs test, the quantitative values of the 5 peptide fragments showed outliers and were significantly smaller than the average quantitative values (Table 6, bold and Underline).
  • NVTLCCLTK SEQ ID NO: 5
  • ILSGGALQLWNVTR SEQ ID NO: 2
  • SSTVSTAEVDPNYYSMR SEQ ID NO: 3
  • the amount of post-translationally modified protein is 1.2 fmol / glomera for NVTLCCLTK (SEQ ID NO: 5), 1.3 fmol / glomera for ILSGGALQLWNVTR (SEQ ID NO: 2), 0.7 fmol / thread for SSTVSTAEVDPNYYSMR (SEQ ID NO: 3)
  • NVTLCCLTK SEQ ID NO: 5
  • ILSGGALQLWNVTR SEQ ID NO: 2
  • ILSGGALQLWNVTR is of the protein that has undergone sugar chain modification is 100.
  • the present invention can be suitably used in the field of protein analysis using a mass spectrometer. Quantitative accuracy, cost, quantification of post-translational modifications, and simplicity are easy, and post-translational modifications and genetic mutation identification and quantification can be performed simultaneously, dramatically reducing the analysis time per protein and simultaneously measuring multiple molecules. It is also useful in the field of analysis and screening, and the field of contract analysis business for protein analysis. In addition, it is a technology that makes it possible to search for unknown post-translational modification sites of proteins and quantify the rate of modification, which has been difficult in the past, and is also useful for analyzing factors related to diseases, such as disease research and drug development. It can also be suitably used in the life science and medical fields.

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Abstract

Les procédés de quantification de protéines très sensibles classiques à l'aide d'un spectromètre souffraient d'un problème de perte d'échantillon et de taux de non-fragmentation lors du procédé de fragmentation de la protéine cible non corrigé par des fragments peptidiques de référence interne. De plus, l'identification et/ou la quantification d'une modification post-traductionnelle, etc., des protéines à l'aide d'un spectromètre étaient extrêmement difficiles. Cette invention concerne, par conséquent, la mise au point d'un procédé de quantification de protéines de haute précision faisant appel à un spectromètre et d'identification et/ou de quantification des modifications post-traductionnelles ou des mutations génétiques survenant dans des protéines. Les présents inventeurs ont découvert qu'une pluralité de fragments peptidiques dérivés de la protéine cible peut être détectée d'un coup à l'aide du spectromètre et que les protéines peuvent être quantifiées à une sensibilité élevée et une précision élevée, à l'aide d'une protéine de référence interne stable, marquée par un isotope. Les présents inventeurs ont également découvert que les modifications post-traductionnelles et les mutations génétiques de chaque fragment peptidique pouvaient être identifiées et/ou quantifiées à l'aide de la valeur de quantification moyenne des fragments peptidiques dérivés de la protéine cible détectés.
PCT/JP2012/000227 2011-02-14 2012-01-17 Procédé de détection d'un changement de masse dans un procédé de spectrométrie de masse et procédé de quantification de la quantité absolue d'une protéine stable marquée par un isotope WO2012111249A1 (fr)

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