CN101042375A - Method and reagent kit for rare-earth element metal chelating making quantitative proteome - Google Patents
Method and reagent kit for rare-earth element metal chelating making quantitative proteome Download PDFInfo
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Abstract
This invention provides to rare earth element metal chelation label protein for quantitative protein study method and agent case. This invention method relates to protein restore alkali base and pancreatic enzymes slices, peptide section double function agent decoration, rare metal chelation, multi-diemension liquid phase spectrum isolation and multiple degrees series spectrum quantitative and testing. Through one degree spectrum ion signal intensity content and protein validating through series mass spectrum and database. This invention also describes this method agent case.
Description
Technical field
The present invention relates to the method and the kit of quantification of protein in a kind of proteomics.Specifically, relate to a kind of new method and kit that is used for the rare-earth element metal chelating labelled protein of quantitative proteomics.
Background technology
Quantitative proteomics is by someway or technology, Protein content in the biological sample in some process (cell, tissue or body fluid etc.) is compared analysis, being on the protein group level accurate quantitative analysis to be carried out in gene expression, is the necessary means and the research forward position of current research major disease mechanism of causing a disease and pharmacology controlling mechanism.
Current, the relative quantification method of widespread use in the proteome research mainly contains and relies on two dimensional gel electrophore-sis (Twodimensional electrophoresis, colouring method 2-DE) and the mass spectrum detection of cold labeling.2-DE relative quantification method can reflect the difference of protein expression intuitively by staining power, but the existence of dyestuff brings interference for easily follow-up Mass Spectrometer Method.In addition, the effect that 2-DE separates is not fine, and the numerous protein point has comprised more than one albumen; And can not be high or extremely low to molecular weight, the isoelectric point protein that extremely acid or utmost point alkali and content are low and memebrane protein etc. effectively separate and present, and therefore can not adapt to the needs that present proteome research deeply develops.
Second kind of quantitative strategies is to introduce the quantitative strategies of stable isotope chemical labeling connexus spectral technology.In recent years, quantitative proteomics mainly is the stable isotope chemical marker method of core with the Mass Spectrometer Method.But be subjected to the synthetic difficulty of isotope reagent, the restriction of factors such as price height.
It is quality tab that people such as Meares C.F. avoid with the stable isotope, and with the very close thulium of chemical property as quality tab, " rubidium marking affinity tag " (ECAT has been proposed, Element-coded affinity tags, WhetstoneP.A., Butlin N.G., Corneillie T.M., and Meares C.F., Bioconjugate Chem., 2004; New method 15:3-6), and synthesized a standard peptide section and verified the chromatogram co-elute of the peptide section potpourri that is marked with rare earth element yttrium and terbium respectively and the response in the mass spectrum.But they can be with this method quantitative problem that solves in the proteomics.
Summary of the invention
The invention provides a kind of is the proteomics relative quantification method of quality tab with the very close rare earth metal of chemical property.The inventor finds in conjunction with multidimensional liquid chromatography separation means and plural serial stage mass spectrum, using price is feasible well below the rare earth element of stable isotope as the quality internal standard in the quantitative proteomics, can be with the relative quantification problem that solves proteomics.
Specifically, the invention provides a kind of method that is used for quantitative rare-earth element metal chelating labelled protein, this method comprises:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carries out mass spectrum quantitative;
The disposal route that it is characterized in that described step (3) is:
(a) with one or more bifunctional chelating agents the peptide section is modified, make it be with chelation group;
(b) use two kinds of different or multiple single isotope rare earth elemental metals salt ions respectively, with the sequestrant generation chelation that is marked on the peptide section;
(c) two kinds of peptide section potpourris that are marked with the sample to be compared of different rare earth ions are respectively merged, will merge the peptide section with one or more dimensions chromatographic resolution means and separate;
(d) the quantitative and evaluation peptide section with mass spectrum is carried out the parsing of mass spectrometric data and the evaluation of protein.
Wherein, the described bifunctional chelating agent of step (a) is preferably band aminopolycanboxylic acid in the molecule, and aromatic amine polycarboxylic acid, or the compound of the active spacer group of isothiocyanic acid Quito carboxylic acid are preferably diethylenetriamine pentaacetic acid bisgallic acid acid anhydride especially.
The described rare earth element of step (b) is preferably yttrium trichloride and terbium trichloride.
The preferred LC-Q-TOF-MS/MS electro-spray ionization of the described mass spectrum of step (d) tandem mass spectrum, or the MALDI TOF/TOF ground substance assistant laser flight time tandem mass spectrum that dissociates more preferably use LC-MS; Database search method is preferably adopted in the parsing of mass spectrometric data and the evaluation of protein.
This method can be carried out the modification of difunctional chelation group to the peptide section that the protein enzyme of two kinds of different physiological statuss is cut generation; Carry out the chelating mark with two kinds of different single isotopic rare earth elemental metals ions again, the peptide section potpourri that will be marked with the different physiological statuss of different rare earth ions then combines, and separates and the tandem mass spectrum assay determination by multi-dimensional chromatograph.The same peptide section that is marked with different rare earth ions can be by co-elute in liquid phase separation, and the right mass spectrum response of the mass spectral quasi-molecular ions of one-level with expect consistent.Mass number phase difference is the ionic strength ratio of a pair of peptide section of the mass number difference of rare earth elemental metals in the one-level mass spectrogram, the relative scale of just corresponding two kinds of comparison proteins, the right mass number difference of unicharged quasi-molecular ions is that the mass number of single isotope rare earth elemental metals is poor, the mass number difference of doubly charged ion pair be single isotope rare earth elemental metals the mass number difference 1/2nd, the rest may be inferred.The peak list file that generates by second order ms carries out database retrieval and comes identification of protein.In one-level mass spectrum response signal, the inventor finds that the peptide section that is marked with terbium metal has ion to suppress phenomenon to the peptide section that is marked with metallic yttrium.When the blending ratio of two kinds of albumen samples setting was 1: 1, mark yttrium metallic ion was 0.65: 1 with the average proportions that is marked with the ionic strength of terbium metal ion peptide section from the one-level mass spectrogram, good reproducibility.And be linear change between preset proportion 1.0 to 10, linear relationship is good, and related coefficient is more than 0.99.
Bifunctional chelating agent is in the molecule or the band aminopolycanboxylic acid, or band aromatic amine polycarboxylic acid, or the compound of the active spacer group of band isothiocyanic acid Quito carboxylic acid.As diethylenetriamine pentaacetic acid bisgallic acid acid anhydride (bicyclic anhydride diethylenetriamine-N, N, N '; N "; N "-pentaacetic dianhydride, DTPAA) be difunctional chelating reagent, its end can with the amido coupling of peptide section, an end can with the rare earth ion chelating.Bifunctional chelating agent is widely used in radiommunoassay and the targeted radiotherapy, relative low price.In addition, the rare earth metal amount is inexpensive greatly, and chemical property is very similar, so it is huge at the application potential of proteomics in quantitatively to be quality tab connexus spectrum detection method with the thulium.
On the other hand, the present invention also provides the kit that is used for quantitative proteomics middle rare earth metal element chelating labelled protein, and the component of this kit comprises:
(a) one or more are modified the peptide section, make it with the bifunctional chelating agent of going up chelation group;
(b) two or more single rare earth element trichlorides;
(c) can carry out the chromatographic column of peptide section chromatographic resolution.
Wherein, the interior or band aminopolycanboxylic acid of the preferred molecule of component (a), aromatic amine polycarboxylic acid, or the compound of the active spacer group of band isothiocyanic acid Quito carboxylic acid; The preferred reversed-phase column of component (c) or ion exchange column, more preferably on-line coupling chromatographic column.
Use for convenient, this kit also can comprise one or more in the following component: be used for opening the reductive agent of protein molecule disulfide bond, as dithiothreitol (DTT) (DTT) and three carboxyethyl phosphines (TCEP); The sealing sulfydryl prevents that it from forming the alkylating reagent of disulfide bond again, as iodoacetamide and iodoacetic acid; The protein digestibility agent is as trypsase; The side chain amido protecting agent is as the O-methyl-isourea; Can contain the buffer system that one or more are used for peptide section chromatographic resolution in addition; The operation instruction of state administrative organs's approval etc.
New method of the present invention and kit be applicable to the protein difference expression to the tissue of two or more different conditions or cell carry out relative quantitatively, this method is quick, and is easy to use.Difference under can more different simultaneously physiological and pathological conditions on the protein expression level of two or more samples, thereby the easy biomarker that screens this physiological and pathological condition of sign rapidly and accurately is a medical diagnosis on disease and drug screening analysis tool simply fast.Quantification of protein and qualitative analysis can be finished in an experimental period synchronously.Be specially adapted to carry out in biology, medical science and the field of pharmacology differential protein group or comparison protein group analysis.
This method is a protein difference expression relative quantitative assay instrument, reduces the cost of relative quantification in the proteomics greatly.And reaction conditions is gentle, simple to operate relatively, realizes easily.Quantification of protein and qualitative analysis can be finished in an experimental period synchronously.Method of the present invention and kit have practicality widely.Purposes includes but not limited to: the proteome research that carries out various animals and plants and microorganism in fields such as medical science, pharmacy, agricultural and animal husbandry.
Description of drawings
Fig. 1 .A figure be insulin GFFYTPK respectively mark the one-level mass spectrogram with the co-elute peptide section of two positive charges of Y and Tb label, the co-elute time is 29.87 minutes, can see that m/z is one group of peak of 660.58 and 695.55, their mass number differs 35Da.B figure is that theoretical ratio is set at 0.5: 1.0 o'clock, and one-level mass spectrogram intermediate ion intensity Y/Tb is 0.38: 1.0.
Fig. 2. insulin pancreatin enzyme the has been cut peptide section FVNQHLCGSHLVEALYLVCGER mark second order ms figure with three positive charge peptide sections of Tb label.
Embodiment
The following examples will be further explained the present invention, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.Some change that those skilled in the art is made within the scope of the claims and adjust also should be thought and belongs to scope of the present invention.
The relative quantification of embodiment 1 standard protein insulin
1.1 get 0.1mg insulin, be dissolved in the bicarbonate triethylamine buffer solution of 100 μ L 50mM pH 7.0.Add 0.2 μ L100mM reductive agent, three carboxyethyl phosphorus (TCEP), 37 ℃ of reactions were cooled to room temperature after 4 hours, added 0.5 μ L 100mM iodoacetamide (IAA), and the reaction of room temperature lucifuge adds 10mM DTT cessation reaction after 1 hour.Add 2ug trypsase, 37 ℃ of enzymes were cut 2 hours.Add 2ug trypsase again, 37 ℃ of enzymes were cut 10 hours.Enzyme is cut in the pressed powder that the insulin peptide section potpourri that obtains joins 360 μ g diethylenetriamine pentaacetic acid bisgallic acid acid anhydrides, and behind the violent whirlpool shake 1min, normal temperature was placed 1 hour.After then this reaction solution being put into the vacuum drier bone dry, add dissolving again in the 0.1M Ammoniom-Acetate solution, be divided into two parts according to volume averaging, it is the YCl of 0.15M that a copy of it adds 40 μ L concentration
3, the TbCl of same concentration of adding and volume in another part
3Aqueous solution.37 ℃ of temperature were incubated 2 hours.Then two kinds of reaction solutions are mixed according to different ratio (1: 1,1: 2,1: 5,1: 10,2: 1,5: 1,10: 1).
Directly on LC-MS (LC-Q-TOF Micro Waters, the U.S.) instrument, carry out determination and analysis 1.2 respectively get the mixed sample of 0.6 μ L different proportion.The analysis condition of LC-Q-TOF Micro is: liquid phase adopts Waters CapLC liquid chromatographic system, uses 2% acetonitrile, and sample on 0.1% formic acid solution, flow velocity are 20 μ L/min, sample desalination 4 minutes on the PepMap C18 pre-column of 320 μ m i.d. * 1mm.Analytical column adopts C18 capillary column (75 μ m i.d. * 15cm, LC Packings).The gradient elution of chromatographic resolution is: solution A (water/acetonitrile is 95/5, and v/v contains 0.1% formic acid); Solution B (water/acetonitrile is 15/85, and v/v contains 0.1% formic acid), shunting back flow velocity is about 200nl/min.Gradient: 4%B---50%B, 45min; 50%B---100%B, 10min; 100%B keeps 10min, gets back to 4%B with 6min again, balance 15min.The liquid phase eluent directly enters the mass spectrometric spraying source that rises (nanoflow source) of receiving of Q-TOF Micro (Waters), mass spectrum is worked under positive ion mode, 80 ℃ of source temperature, cone gas nitrogen flow rate 50L/h, kapillary is received and risen shower nozzle voltage is 3.2kV, sheath atmospheric pressure 5psi, detecting device MCP voltage 2850V.The signals collecting mode (survey scan) that adopts data to rely on, signal threshold value is made as 7counts, once select 3 or 5 ions that abundance is the highest to carry out the tandem mass spectrum data collection automatically, one-level MS sweep limit m/z (400-1500), 1 second/scan, secondary MSMS sweep limit m/z (80-1500) or m/z (80-2000), 1 second/scan, secondary collision voltage is adjusted automatically according to the electrically charged situation and the specific charge size of parent ion, and MS and MS/MS data obtain under continuous mode.All data MassLynx 4.0 software processes.All MSMS data generate data file peak list and are used for the database retrieval evaluation.Search parameter comprise variable N end that is modified at the peptide section or lysine mark the mass number of DTPA-Y label add 464Da, at the N of peptide section end mark the mass number of DTPA-Tb label add 534Da.
1.3 can see m/z on the one-level mass spectrogram is one group of peak of 660.58 and 695.55, their mass number differs 35Da, for insulin GFFYTPK respectively mark the co-elute peptide section with two positive charges of Y and Tb label.See Figure of description 1.The ratio that is marked with Y and Tb in these peptide sections is 0.65 times of setting value.The another one pancreatin enzyme of insulin cut peptide section FVNQHLCGSHLVEALYLVCGER mark Tb label with the earphone mass spectrogram of three positive charge peptide sections shown in Figure of description 2.
The white relative quantification of embodiment 2 standard protein horses cardiac muscle red eggs
2.1 it is white to get 0.1mg horse cardiac muscle red eggs, is dissolved in the bicarbonate triethylamine buffer solution of 100 μ L 50mM pH 7.0.There is not cysteine residues in the amino acid sequence of myoglobins, so do not need the reductive alkylation step.Add 2ug trypsase, 37 ℃ of enzymes were cut 2 hours.Add 2ug trypsase again, 37 ℃ of enzymes were cut 10 hours.Enzyme is cut in the pressed powder that the myoglobins peptide section potpourri that obtains joins 360 μ g diethylenetriamine pentaacetic acid bisgallic acid acid anhydrides, and behind the violent whirlpool shake 1min, normal temperature was placed 1 hour.After then this reaction solution being put into the vacuum drier bone dry, add dissolving again in the 0.1M Ammoniom-Acetate solution, be equally divided into two parts, it is the YCl of 0.15M that a copy of it adds 40 μ L concentration
3, the TbCl of same concentration of adding and volume in other portion
3Aqueous solution.37 ℃ of temperature were incubated 2 hours.(1: 1,1: 2,1: 5,1: 10,2: 1,5: 1,10: 1 ratio was mixed according to different ratios with two kinds of reaction solutions then.
Directly on the LC-Q-TOF of LC-MS Micro (Waters, the U.S.) instrument, carry out determination and analysis 2.2 respectively get the mixed sample of 0.6 μ L different proportion.The analysis condition of LC-Q-TOF Micro is: liquid phase adopts Waters CapLC liquid chromatographic system, uses 2% acetonitrile, and sample on 0.1% formic acid solution, flow velocity are 20 μ L/min, sample desalination 4 minutes on the PepMap C18 pre-column of 320 μ m i.d. * 1mm.Analytical column employing C18 capillary column (75 μ m i.d. * 15cm, LCPackings).The gradient elution of chromatographic resolution is: solution A (water/acetonitrile is 95/5, and v/v contains 0.1% formic acid); Solution B (water/acetonitrile is 15/85, and v/v contains 0.1% formic acid), shunting back flow velocity is about 200nl/min.Gradient: 4%B---50%B, 45min; 50%B---100%B, 10min; 100%B keeps 10min, gets back to 4%B with 6min again, balance 15min.The liquid phase eluent directly enters the mass spectrometric spraying source that rises (nanoflow source) of receiving of Q-TOF Micro (Waters), mass spectrum is worked under positive ion mode, 80 ℃ of source temperature, cone gas nitrogen flow rate 50L/h, kapillary is received and risen shower nozzle voltage is 3.2kV, sheath atmospheric pressure 5psi, detecting device MCP voltage 2850V.The signals collecting mode (survey scan) that adopts data to rely on, signal threshold value is made as 7counts, once select 3 or 5 ions that abundance is the highest to carry out the tandem mass spectrum data collection automatically, one-level MS sweep limit m/z (400-1500), 1 second/scan, secondary MSMS sweep limit m/z (80-1500) or m/z (80-2000), 1 second/scan, secondary collision voltage is adjusted automatically according to the electrically charged situation and the specific charge size of parent ion, and MS and MS/MS data obtain under continuous mode.All data MassLynx 4.0 software processes.All MSMS data generate data file peak list and are used for the database retrieval evaluation.Search parameter comprise variable N end that is modified at the peptide section or lysine mark the mass number of DTPA-Y label add 464Da, at the N of peptide section end mark the mass number of DTPA-Tb label add 534Da.
2.3 can see m/z on the one-level mass spectrogram is one group of peak of 1034.20 and 1069.25, their mass number differs 35Da, for myoglobins peptide section VEADIAGHGQEVLIR respectively mark the co-elute peptide section with two positive charges of Y and Tb label.In addition the m/z that can also see is one group of peak of 857.71 and 892.73, for myoglobins peptide section TEAEMK respectively mark the co-elute peptide section with two positive charges of Y and Tb label, or the like.The ratio that is marked with Y and Tb in these peptide sections is 0.65 times of setting value.
The relative quantification of 3 six kinds of standard protein potpourris of embodiment
3.1 dispose the concentration of every kind of albumen is six kinds of protein solutions (bovine serum albumin(BSA), transferrins, α-milk-globule albumin, β-milk-globule albumin, myoglobins and bacteriolyze ferment albumen) of 0.1mmoL/L, and buffer solution is the bicarbonate triethylamine of 50mM pH 7.0.Respectively getting 70 μ L mixes.Add 0.8 μ L 500mM reductive agent, three carboxyethyl phosphorus (TCEP), 37 ℃ of reactions were cooled to room temperature after 4 hours, added 1 μ L 1M iodoacetamide (IAA), and the reaction of room temperature lucifuge is after 1 hour.Add 5ug trypsase, 37 ℃ of enzymes were cut 2 hours.Add 5ug trypsase again, 37 ℃ of enzymes were cut 10 hours.Enzyme is cut in the pressed powder that the myoglobins peptide section potpourri that obtains joins 360 μ g diethylenetriamine pentaacetic acid bisgallic acid acid anhydrides, and behind the violent whirlpool shake 1min, normal temperature was placed 1 hour.After then this reaction solution being put into the vacuum drier bone dry, again dissolving in the 0.1M Ammoniom-Acetate solution of adding equivalent, be equally divided into two parts, it is the YCl3 of 0.15M that a copy of it adds 168 μ L concentration, adds the TbCl3 aqueous solution of same concentration and volume in other portion.37 ℃ of temperature were incubated 2 hours.Then two kinds of reaction solutions are mixed according to 1: 1 ratio of different ratios.
Directly on the LC-Q-TOF of LC-MS Micro (Waters, the U.S.) instrument, carry out determination and analysis 3.2 respectively get the mixed sample of 0.6 μ L different proportion.The analysis condition of LC-Q-TOF Micro is: liquid phase adopts Waters CapLC liquid chromatographic system, uses 2% acetonitrile, and sample on 0.1% formic acid solution, flow velocity are 20 μ L/min, sample desalination 4 minutes on the PepMap C18 pre-column of 320 μ m i.d. * 1mm.Analytical column adopts C18 capillary column (75 μ m i.d. * 15cm, LC Packings).The gradient elution of chromatographic resolution is: solution A (water/acetonitrile is 95/5, and v/v contains 0.1% formic acid); Solution B (water/acetonitrile is 15/85, and v/v contains 0.1% formic acid), shunting back flow velocity is about 200nl/min.Gradient: 4%B---50%B, 45min; 50%B---100%B, 10min; 100%B keeps 10min, gets back to 4%B with 6min again, balance 15min.The liquid phase eluent directly enters Q-TOF Micro (the Waters)-mass spectrometric spraying source that rises-(nanoflow source) that receive, mass spectrum is worked under positive ion mode, 80 ℃ of source temperature, cone gas nitrogen flow rate 50L/h, kapillary is received and risen shower nozzle voltage is 3.2kV, sheath atmospheric pressure 5psi, detecting device MCP voltage 2850V.The signals collecting mode (survey scan) that adopts data to rely on, signal threshold value is made as 7counts, once select 3 or 5 ions that abundance is the highest to carry out the tandem mass spectrum data collection automatically, one-level MS sweep limit m/z (400-1500), 1 second/scan, secondary MSMS sweep limit m/z (80-1500) or m/z (80-2000), 1 second/scan, secondary collision voltage is adjusted automatically according to the electrically charged situation and the specific charge size of parent ion, and MS and MS/MS data obtain under continuous mode.All data MassLynx 4.0 software processes.All MSMS data generate data file peak list and are used for the database retrieval evaluation.Search parameter comprise variable N end that is modified at the peptide section or lysine mark the mass number of DTPA-Y label add 464Da, at the N of peptide section end mark the mass number of DTPA-Tb label add 534Da.
3.3 in the result of mass spectroscopy, all MSMS data generate data file peak list and have identified this six standard proteins by database retrieval, and the average ion strength ratio of the one group of group peptide section that is marked with Y and Tb label that electrically charged number is different in their one-level mass spectrum is 0.65.It is 0.65 times of preset proportion 1.
Claims (10)
1. method that is used for quantitative rare-earth element metal chelating labelled protein, this method comprises:
(1) opens disulfide bond in the protein molecule with reductive agent, destroy its higher structure; Seal sulfydryl with alkylating reagent, prevent that it from forming disulfide bond again;
(2) with chemical digestion or enzyme digestion protein is digested, produce the peptide section that is suitable for mass spectrophotometry; Protect with the side chain amino that reagent is cut the peptide section to enzyme;
(3) the peptide section is carried out chemical modification and processing and carries out mass spectrum quantitative;
The disposal route that it is characterized in that described step (3) is:
(a) with one or more bifunctional chelating agents the peptide section is modified, make it be with chelation group;
(b) use two kinds of different or multiple single isotope rare earth elemental metals salt ions respectively, with the sequestrant generation chelation that is marked on the peptide section;
(c) two kinds of peptide section potpourris that are marked with the sample to be compared of different rare earth ions are respectively merged, will merge the peptide section with the chromatographic resolution means and separate;
(d) the quantitative and evaluation peptide section with mass spectrum is carried out the parsing of mass spectrometric data and the evaluation of protein.
2. the method for claim 1 is characterized in that the described bifunctional chelating agent of step (a) is band aminopolycanboxylic acid in the molecule, aromatic amine polycarboxylic acid, or the compound of the active spacer group of isothiocyanic acid Quito carboxylic acid.
3. the method for claim 1 is characterized in that the described bifunctional chelating agent of step (a) is a diethylenetriamine pentaacetic acid bisgallic acid acid anhydride.
4. the method for claim 1 is characterized in that the described rare earth element of step (b) is yttrium trichloride and terbium trichloride.
5. the method for claim 1 is characterized in that the described mass spectrum of step (d) is a LC-ESI-MS/MS electro-spray ionization tandem mass spectrum, or the MALDI TOF/TOF ground substance assistant laser flight time tandem mass spectrum that dissociates.
6. the method for claim 1 is characterized in that the parsing of the described mass spectrometric data of step (d) and the authentication method of protein are the use database search.
7. the method for claim 1 is characterized in that the disposal route of described step (3) is:
(a) with diethylenetriamine pentaacetic acid bisgallic acid acid anhydride the peptide section is modified, make it be with chelation group;
(b) respectively with yttrium trichloride and terbium trichloride and be marked at sequestrant generation chelation on the peptide section;
(c) two kinds of peptide section potpourris that are marked with the sample to be compared of different rare earth ions are respectively merged, will merge the peptide section with one or more dimensions chromatographic resolution means and separate;
(d) with ground substance assistant laser dissociate the flight time tandem mass spectrum quantitatively with identify the peptide section, use database search to carry out the parsing of mass spectrometric data and the evaluation of protein.
8. kit that is used for quantitative proteomics middle rare earth metal element chelating labelled protein is characterized in that the component of this kit comprises:
(a) one or more are modified the peptide section, make it with the bifunctional chelating agent of going up chelation group;
(b) two or more single rare earth element trichlorides;
(c) can carry out the chromatographic column of peptide section chromatographic resolution.
9. kit as claimed in claim 8 is characterized in that the described bifunctional chelating agent of component (a) is in the molecule or the band aminopolycanboxylic acid, aromatic amine polycarboxylic acid, or the compound of the active spacer group of band isothiocyanic acid Quito carboxylic acid.
10. kit as claimed in claim 8 is characterized in that the described chromatographic column of component (c) is reversed-phase column or ion exchange column.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105137088A (en) * | 2015-08-27 | 2015-12-09 | 同济大学 | Whole-body-protein quantitative analysis method |
| CN105259118A (en) * | 2015-10-15 | 2016-01-20 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Difunctional nanoprobe based on lanthanide metal as well as preparation method and application of difunctional nanoprobe |
| CN117491653A (en) * | 2023-11-06 | 2024-02-02 | 上海体育大学 | Preparation method of growth hormone sample |
-
2006
- 2006-03-20 CN CN 200610065077 patent/CN101042375A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105137088A (en) * | 2015-08-27 | 2015-12-09 | 同济大学 | Whole-body-protein quantitative analysis method |
| CN105259118A (en) * | 2015-10-15 | 2016-01-20 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Difunctional nanoprobe based on lanthanide metal as well as preparation method and application of difunctional nanoprobe |
| CN105259118B (en) * | 2015-10-15 | 2019-03-08 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal |
| CN117491653A (en) * | 2023-11-06 | 2024-02-02 | 上海体育大学 | Preparation method of growth hormone sample |
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