CN106770821A - The method that peptide fragment isotope dilution mass spectrometry quantitatively recombinates Troponin I content - Google Patents
The method that peptide fragment isotope dilution mass spectrometry quantitatively recombinates Troponin I content Download PDFInfo
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Abstract
The method that peptide fragment isotope dilution mass spectrometry quantitatively recombinates Troponin I content, it is related to a kind of method that accurate quantitative analysis recombinate Troponin I content；Comprise the following steps：Feature peptide fragment is designed；The determination of feature peptide fragment mass spectral analysis ion pair；LC MRM condition researchs；Sample analysis pre-treatment：Key step has：Recombinant protein denaturation, reductive alkylation, enzymolysis, purification of samples, concentrating sample；Data analysis：CTnI specificity peptide fragment concentration, cTnI protein peptides appearance times 4.20min；The rate of recovery and precision：The rate of recovery and precision meet regulation requirement, and restructuring cTnI specificity peptide fragment liquid chromatography mass spectrometric conditions are studied and improved, and institute's construction method degree of accuracy is good, and speed is fast, and appearance time is 4.20min.
Description
Technical field
The present invention relates to a kind of method that accurate quantitative analysis recombinate Troponin I content, and in particular to a kind of peptide fragment isotope
The method that dilution mass spectrum standard measure recombinates Troponin I content.
Background technology
Troponin (cTnI) is one of most sensitive biological indicator of detection myocardial damage.However, cTnI detections are still deposited
In some problems, mainly detection method is not yet standardized, and there is certain difference between distinct methods, and testing result does not usually have
There is comparativity spanning space-time.
Isotope Liquid Chromatography-Tandem Mass Spectrometry (Liquid chromatography-tandem mass
Spectrometry, LC/MS/MS) advantage be high sensitivity, high precision, high degree of specificity, the standardization of its measurement result can
Directly trace to the source to international base unit system.Domestic scholars [1] set up amino acid isotope dilution mass spectrometry and quantitatively recombinate cTnI, should
Method needs pyrohydrolysis amino acid, there is potential safety hazard.
Domestic relevant peptide fragment isotope dilution mass spectrometry Quantitative Western reported containing quantifier elimination it is less, mainly and protein sample
Complicated component, its degree of ionization are easily influenceed by other impurities ion, and experiment condition is complex.
Bibliography：
[1] absolute quantification method [D] north of the fine important biomolecules marks of Sun Xue-alpha-fetoprotein, leptin and Troponin I
Capital university of chemical technology, 2014.
[2] Kuhn E, Addona T, Keshishian H, et al.Developing multiplexed assays
for troponin I and interleukin-33in plasma by peptide immunoaffinity
Enrichment and targeted mass spectrometry.Clin Chem [J] .2009,55 (6)：1108-17.
[3] Keshishian H, Addona T, Burgess M, et al..Quantification of
cardiovascular biomarkers in patient plasma by targeted mass spectrometry and
Stable isotope dilution.Mol Cell Proteomics [J] .2009,8 (10)：2339-49.
[4] Huillet C, Adrait A, Lebert D, et al.Accurate quantification of
cardiovascular biomarkers in serum using Protein Standard Absolute
Quantification(PSAQTM)and selected reaction monitoring[J].Molecular&Cellular
Proteomics Mcp, 2012,11 (2)：8681-8696.
[5] Zhao C, Trudeau B, Xie H, et al.Epitope mapping and targeted
quantitation of the cardiac biomarker troponin by SID-MRM mass
Spectrometry.Proteomics [J] .2014,14 (11)：1311-21.
The content of the invention
Restructuring cTnI specificity peptide fragment liquid chromatography mass spectrometric conditions are studied and improved it is an object of the invention to provide one kind,
Institute's construction method degree of accuracy is good, and speed is fast, and appearance time quantitatively recombinates flesh calcium egg for the peptide fragment isotope dilution mass spectrometry of 4.20min
The method of white I contents.
In order to solve the problems existing in background technology, the present invention is to use following technical scheme：A kind of peptide fragment isotope
The method that dilution mass spectrum standard measure recombinates Troponin I content, comprises the following steps：
(1) feature peptide fragment design：In Pubmed Protein database search people source Troponin I amino acid sequences,
Accession：NP_000354.4, then on-line analysis mass spectrum peptide fragment, network address：http：//web.expasy.org/
Peptide_mass/, Binding experiment room mass spectrometer feature selection purpose peptide fragment, then by mesh peptide fragment in pubmed albumen prime numbers
Blast Homology search is carried out according to storehouse, is defined as special peptide fragment.
(2) determination of feature peptide fragment mass spectral analysis ion pair：The parent ion of special peptide fragment is studied with skyline software analysis
And daughter ion, the regulation strong three pairs of ions of collision energy selection signal study, and it is quota ion pair that signal is most strong, other
It is qualitative ion pair.
(3) LC-MRM conditions research：Ultrahigh pressure liquid phase chromatogram (UPLC LC-30A) parameter liquid chromatograph：Shimadzu imitates liquid
Phase chromatogram infusion pump and automatic sampler；Chromatographic column：CSH 130C18；40 DEG C of column temperature, mobile phase A：0.1% aqueous formic acid,
Mobile phase B：0.1% formic acid acetonitrile solution, flow velocity 0.3mL/min, Mobile phase B 2min 5%, 8min 50%, 9min 90%,
14min 5%.Mass spectrum separation condition, instrument AB 5500, ion injection electric 5500V；550 DEG C of temperature；Curtain gas (CUR)
It is 25L/min, collision gas (CAD) 8L/min；Positive ion mode is detected；Scan mode is multiple-reaction monitoring (MRM).
(4) sample analysis pre-treatment：Key step has：It is recombinant protein denaturation, reductive alkylation, enzymolysis, purification of samples, dense
Contracting sample.
(5) data analysis：With in software Analystl.2.1 analytical standards peptide (ion pair 623.3/1018.6)/isotope
Mark peptide (ion pair 626.8/1025.6) peak area simultaneously makes standard curve.CTnI peptide fragment concentration is calculated according to standard curve.Weight
Histone cTnI concentration (ng/mL) is that cTnI specificity peptide fragment concentration is multiplied by 19.28；19.28 by cTnI molecular weight (24016Da)
Divided by cTnI features peptide molecular weight (1245.41Da).
(6) rate of recovery and precision：It is 62.1ng/mL, 621ng/mL aqueous solution to prepare cTnI contents, before sample analysis
Treatment and measure, the ratio between measured value and theoretical value are the method rate of recovery.Meanwhile, two concentration protein samples low, high are respectively at 1d
Interior METHOD FOR CONTINUOUS DETERMINATION 5 times, it is continuous to survey 5 days, calculate batch interior and betweenrun precision.
As a further improvement on the present invention；The concrete operation method of described step (1) is：According to people source cTnI albumen
Matter amino acid sequence, cTnI specificity peptide fragments are surveyed with Peptide mass Software for Design isotope dilution mass spectrometry
NITEIADLTQK, it is specific peptide fragment to retrieve the peptide fragment through BLAST, and theoretical molecular is 1245.7Da；Synthesizing peptide molecular weight is
1245.41Da, predominantly divalent charge in the solution, molecular weight is 623.3Da；In specific peptide fragment L amino acid sites mark
Isotope (13C6 15N), label isotope molecular weight is 7Da, and isotope marks peptide fragment molecular weight is 1252.6Da, is led in the solution
To be divalent charge, molecular weight is 626.8Da；The triple specific peptide fragment ion pairs of level Four bar mass spectrograph identification of instrument AB 5500
It is 623.3/1018.6,623.3/675.1,623.3/788.6；Isotope marks peptide fragment ion pair be 626.8/1025.6,
626.8/682.1,626.8/795.5, wherein 623.3/1018.6 are absolute quantitation ion pair with 626.8/1025.6.
As a further improvement on the present invention；In described step (4), sample analysis pre-treatment is concretely comprised the following steps：
I) it is denatured, 25 μ L protein liquids samples add the μ L of 1ng/mL isotope marks peptide 7.5, then add 217.5 μ L 25mmol/L
Ammonium hydrogen carbonate is mixed, plus urea 96mg makes its final concentration of 6M, is mixed, and stands 5min；
II) reductive alkylation, plus 250 μ L 25mmol/L ammonium hydrogen carbonate, plus 100mM dithiothreitol (DTT)s (DTT) 50 μ L, 60
DEG C water-bath 30min.Alkylation, plus the μ L of 550mM iodoacetamides (IAA) 50.Room temperature lucifuge 20min.Plus the μ L of 200mM DTT 50, room
Warm 20min.Plus the μ L of 25mmol/L ammonium hydrogen carbonate 1000, urea concentration is less than 1M；
III) digest, add pancreatin, pancreatin and sample quality ratio are 1: 10,37 DEG C of water-bath 4h, plus 0.1% trifluoroacetic acid
(TFA) 30 μ L terminating reactions；
IV) purification of samples, plus 3.0mL methyl alcohol activates OASIS MAX solid-phase extraction columns (3cc/60 μm), after methyl alcohol flows to end
Plus 3.0mL deionizations balance pillar, plus enzymolysis product is in post, is successively washed with 2.0mL10% ammoniacal liquor, 2.0mL methyl alcohol, finally
Eluted with the formic acid methyl alcohol of 2.0mL 2%；
V) concentrating sample, the concentrate eluant on Nitrogen evaporator, gas flow 5mL/min, temperature 45 C treats that organic solvent is waved
After hair, dissolved with 0.1mL 0.1%FA.Upper machine carries out liquid chromatography mass spectrometric detection, the μ L of sample size 5.
As a further improvement on the present invention；In described step (5), the specific method of data analysis is：
CTnI specificity peptide fragment concentration, cTnI protein peptides appearance time 4.20min are analyzed with software Analyst1.2.1 and marked
Quasi- peptide (ion pair 623.3/1018.6)/Isotopic Internal Standard peptide (ion pair 626.8/1025.6) peak area simultaneously makes standard song
Line, weighted line regress equation (" 1/x " weighting)：Y=0.208x-0.072 (r=0.9988), according to standard curve meter
Calculate cTnI peptide fragment concentration；It is prepared by cTnI peptides standard curve：Standard peptide amino acid isotope dilution mass spectrometry is traced to the source SI units, will
It uses mass spectrum level water to prepare various concentrations 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, respectively
The μ L of 1ng/mL isotope marks peptide 7.5 are added to mix, detection pretreatment process is identical with the step (4) of step (1).
As a further improvement on the present invention；In described step (6), content is that the suitable peptide of the 62.1ng/mL aqueous solution is dense
The suitable peptide concentration 32.2ng/mL of degree 3.22ng/mL, 621ng/mL aqueous solution；CTnI protein recoveries low, high are respectively
94.65% and 103.15%.CTnI is low, sample betweenrun precision (CV) high is respectively 7.04%, 5.69%, withinrun precision
(CV) it is respectively 6.15%, 4.76%.The rate of recovery and precision meet regulation requirement.
After adopting the above technical scheme, the invention has the advantages that：
CTnI specificity peptide fragments are filtered out according to Pubmed databases, peptide fragment isotope dilution mass spectrometry is set up and is quantitatively recombinated
The method of cTnI.This method is significantly different fixed with amino acid isotope dilution mass spectrometry at aspects such as standard items, Isotopic Internal Standards
Amount restructuring Troponin I.This law testing result can equally trace to the source to SI.For research serum cTnI reference material Quality Research establishes base
Plinth.
Brief description of the drawings
Fig. 1 is the selection figure of the bioinformatic analysis specificity peptide fragment of embodiment provided by the present invention；
In http：//web.expasy.org/peptide_mass/ databases carry out the analysis of cTnI pancreatin restriction enzyme site,
Fig. 1 is that cTnI pancreatin restriction enzyme site unit price and divalence predict the outcome, and with reference to mass spectrometer feature, square frame is final choice result；
Fig. 2 is two grades of scanning figures of specific peptide fragment ion massspectrum of embodiment provided by the present invention；
Fig. 3 is two grades of scanning figures of peptide fragment ion massspectrum of the isotope marks of embodiment provided by the present invention；
Fig. 4 is the cTnI LC-MS/MS figures of embodiment provided by the present invention.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with specific embodiment
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only used to explain the present invention, and
It is not used in the restriction present invention.
This specific embodiment uses following technical scheme：A kind of peptide fragment isotope dilution mass spectrometry quantitatively recombinates flesh calcium egg
The method of white I contents, comprises the following steps：
(1) peptide fragment design：
According to people source cTnI protein amino acid sequences, surveyed with Peptide mass Software for Design isotope dilution mass spectrometry
CTnI specificity peptide fragment NITEIADLTQK, are shown in Fig. 1, and it is specific peptide fragment to retrieve the peptide fragment through BLAST, and theoretical molecular is
1245.7Da.Synthesis peptide molecular weight is 1245.41Da, in the solution predominantly divalent charge, and molecular weight is 623.3Da.We
Specific peptide fragment L amino acid sites label isotope (13C6 15N), label isotope molecular weight is 7Da, isotope marks peptide fragment
Molecular weight is 1252.6Da, in the solution predominantly divalent charge, and molecular weight is 626.8Da.This specific embodiment marks peptide
Section is different from external report, is specifically shown in Table 1：
The special peptides of table 1cTnI and isotope marks peptide compare
The triple level Four bar mass spectrographs of instrument AB 5500 identify that specific peptide fragment ion pair is 623.3/1018.6,623.3/
675.1、623.3/788.6.Isotope marks peptide fragment ion pair is 626.8/1025.6,626.8/682.1,626.8/795.5.
Result is shown in Fig. 2, Fig. 3, and wherein 623.3/1018.6 and 626.8/1025.6 is absolute quantitation ion pair.
(2) liquid phase separation condition：
Ultrahigh pressure liquid phase chromatogram (UPLC LC-30A) parameter liquid chromatograph：Shimadzu effect liquid phase chromatogram infusion pump and automatic
Injector (Japanese firm)；Chromatographic column：Water companies CSH 130C18 (150mm × 2.1,3.5 μm, Waters, US)；
40 DEG C of column temperature, mobile phase A：0.1% aqueous formic acid, Mobile phase B：0.1% formic acid acetonitrile solution, flow velocity 0.3mL/min, flowing
Phase B：2min 5%, 8min 50%, 9min 90%, 14min 5%.
(3) mass spectrum separation condition：
Instrument AB 5500, ion injection electric 5500V；550 DEG C of temperature；Curtain gas (CUR) are 25L/min, collision gas
Body (CAD) 8L/min；Positive ion mode is detected；Scan mode is multiple-reaction monitoring (MRM).Design parameter is shown in Table 2：
The mass spectrum optimal conditions of table 2
(4) sample analysis pre-treatment：
Key step has：Recombinant protein denaturation, reductive alkylation, enzymolysis, purification of samples, concentrating sample.
A) it is denatured, 25 μ L protein liquids samples add the μ L of 1ng/mL isotope marks peptide 7.5, then add 217.5 μ L 25mmol/L
Ammonium hydrogen carbonate is mixed, plus urea 96mg makes its final concentration of 6M, is mixed, and stands 5min.
B) reductive alkylation, plus 250 μ L 25mmol/L ammonium hydrogen carbonate, plus 100mM dithiothreitol (DTT)s (DTT) 50 μ L, 60 DEG C
Water-bath 30min.Alkylation, plus the μ L of 550mM iodoacetamides (IAA) 50.Room temperature lucifuge 20min.Plus the μ L of 200mM DTT 50, room temperature
20min.Plus the μ L of 25mmol/L ammonium hydrogen carbonate 1000, urea concentration is less than 1M.
C) digest, add pancreatin, pancreatin and sample quality ratio are 1: 10,37 DEG C of water-bath 4h, plus 0.1% trifluoroacetic acid
(TFA) 30 μ L terminating reactions.
D) purification of samples, plus 3.0mL methyl alcohol activates OASIS MAX solid-phase extraction columns (3cc/60 μm), after methyl alcohol flows to end
Plus 3.0mL deionizations balance pillar, plus enzymolysis product is in post, is successively washed with 2.0mL10% ammoniacal liquor, 2.0mL methyl alcohol, finally
Eluted with the formic acid methyl alcohol of 2.0mL 2%.
E) concentrating sample, the concentrate eluant on Nitrogen evaporator, gas flow 5mL/min, temperature 45 C treats that organic solvent is waved
After hair, dissolved with 0.1mL 0.1%FA.Upper machine carries out liquid chromatography mass spectrometric detection, the μ L of sample size 5.
(5) prepared by cTnI peptides standard curve：Standard peptide amino acid isotope dilution mass spectrometry is traced to the source SI units.Used
Mass spectrum level water prepares various concentrations 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, is separately added into
The μ L of 1ng/mL isotope marks peptide 7.5 are mixed.Detection pretreatment process is identical with step 1-4.
(6) data analysis：
A) cTnI specificity peptide fragment concentration, cTnI protein peptides appearance time 4.20min are shown in Fig. 4.Use software
Analystl.2.1 analytical standards peptide (ion pair 623.3/1018.6)/Isotopic Internal Standard peptide (ion pair 626.8/1025.6) peak
Area simultaneously makes standard curve.Weighted line regress equation (" 1/x " weighting)：Y=0.208x-0.072 (r=
0.9988).CTnI peptide fragment concentration is calculated according to standard curve.
B) recombinant protein cTnI concentration (ng/mL) is equal to cTnI specificity peptide fragment concentration and is multiplied by 19.28.Note：19.28 by
CTnI molecular weight (24016Da) is divided by cTnI features peptide molecular weight (1245.41Da).
(7) rate of recovery and precision
It is 62.1ng/mL (suitable peptide concentration 3.22ng/mL), 621ng/mL (suitable peptide concentrations to prepare cTnI contents
32.2ng/mL) the aqueous solution, by sample analysis pre-treatment and measure, the ratio between measured value and theoretical value are the method rate of recovery.Meanwhile,
Two concentration protein samples low, high continuously survey 5d respectively at METHOD FOR CONTINUOUS DETERMINATION in 1d 5 times, calculate batch in and betweenrun precision.Low,
CTnI protein recoveries high are respectively 94.65% and 103.15%.CTnI is low, sample betweenrun precision (CV) high is respectively
7.04%th, 5.69%, withinrun precision (CV) is respectively 6.15%, 4.76%.The rate of recovery and precision meet regulation requirement.
This specific embodiment is with the difference of prior art：
1st, the peptide fragment of selection is representative, is cTnI specific peptides.Finally determine purpose using bioinformatics technique
Peptide fragment NITEIADLTQK.It is consistent [2-4] with foreign scholar's report.When detecting polypeptide using UPLC-MS methods, the response letter of polypeptide
Number increase or suppress with instrument and react chemical reagent it is relevant.Kuhn E etc. [2] and Keshishian H [3] have synthesized together
The special peptide NITEIAD of position element [(13C6), L] TQK, the 6Da more than standard peptide.Huillet C [4] have synthesized the special peptide of isotope
NITEIADLTQ[(13C6 15N2) K], the 8Da more than standard peptide.The special peptide NITEIAD of isotope is synthesized with reference to characteristic amino acid
[(13C6 15N) L] TQK, 7Da (being shown in Table 1) more than standard peptide NITEIADLTQK.The physicochemical property and target of the special peptide of the isotope
Polypeptide is completely the same, and the chromatographic mass spectrometry behavior for being showed is also consistent, can correct the detection brought by matrix effect and respond
Value fluctuation.
2.LC-MS/MS results, detecting system is AB Triple quad 5500LC/MS/MS, and the systemic resolution is good,
Sensitive height, mass spectrum optimal conditions are shown in Table 2.Kuhn E AB 4000Q TRAP LC/MS/MS detecting systems, testing conditions and sheet
Basically identical in specific embodiment, Huillet C API5500Q-Trap detecting systems, document does not provide mass spectrum optimization
Condition.Kuhn E report ion pair 623.3/1018.5 signals are most strong, 20min appearances.Huillet C report ion pair 623.3/
675.4 signals are most strong, and 623.3/1018.5 takes second place, appearance time 36.2min, the ion pair 623.3/ of this specific embodiment
1018.6 signals are most strong, appearance time 4.20min.It is bent that Zhao C etc. [5] set up another specificity peptide fragment solution standards of cTnI
Line, standard curve lower limit 4ng/mL, the peptide solution standard curve lower limit that this specific embodiment is set up is 1ng/mL.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each implementation method is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Specification an as entirety, the technical scheme in each embodiment can also be formed into those skilled in the art through appropriately combined
May be appreciated other embodiment.
Claims (5)
1. the method that peptide fragment isotope dilution mass spectrometry quantitatively recombinates Troponin I content, it is characterised in that including following step
Suddenly：
(1) feature peptide fragment design：In Pubmed Protein database search people source Troponin I amino acid sequences,
Accession：NP_000354.4, then on-line analysis mass spectrum peptide fragment, network address：http：//web.expasy.org/
Peptide_mass/, Binding experiment room mass spectrometer feature selection purpose peptide fragment, then by mesh peptide fragment in pubmed albumen prime numbers
Blast Homology search is carried out according to storehouse, is defined as special peptide fragment；
(2) determination of feature peptide fragment mass spectral analysis ion pair：The parent ion and son of special peptide fragment are studied with skyline software analysis
Ion, the regulation strong three pairs of ions of collision energy selection signal are studied, and it is quota ion pair that signal is most strong, and other are fixed
Property ion pair；
(3) LC-MRM conditions research：Ultrahigh pressure liquid phase chromatographic parameter liquid chromatograph：Shimadzu effect liquid phase chromatogram infusion pump and automatic
Injector；Chromatographic column：CSH130C18；40 DEG C of column temperature, mobile phase A：0.1% aqueous formic acid, Mobile phase B：0.1% formic acid second
Nitrile solution, flow velocity 0.3mL/min, Mobile phase B 2min5%, 8min50%, 9min90%, 14min5%；Mass spectrum separation condition,
Instrument AB5500, ion injection electric 5500V；550 DEG C of temperature；Curtain gas are 25L/min, collision gas 8L/min；Just from
Submode is detected；Scan mode is multiple-reaction monitoring；
(4) sample analysis pre-treatment：Key step has：Recombinant protein denaturation, reductive alkylation, enzymolysis, purification of samples, concentration sample
Product；
(5) data analysis：With software Analyst1.2.1 analytical standards peptide/Isotopic Internal Standard peptide peak area and to make standard bent
Line,；CTnI peptide fragment concentration is calculated according to standard curve；Recombinant protein cTnI concentration ng/mL is that cTnI specificity peptide fragment concentration multiplies
With 19.28；19.28 by cTnI molecular weight 24016Da divided by cTnI feature peptide molecular weights 1245.41Da；
(6) rate of recovery and precision：It is 62.1ng/mL, 621ng/mL aqueous solution to prepare cTnI contents, by sample analysis pre-treatment
And measure, the ratio between measured value and theoretical value are the method rate of recovery；Meanwhile, two concentration protein samples low, high in 1d respectively at connecting
Continuous measure 5 times, it is continuous to survey 5 days, calculate batch interior and betweenrun precision.
2. the method that peptide fragment isotope dilution mass spectrometry according to claim 1 quantitatively recombinates Troponin I content, it is special
Levy and be, the concrete operation method of described step (1) is：According to people source cTnI protein amino acid sequences, Peptide is used
Mass Software for Design isotope dilution mass spectrometry surveys cTnI specificity peptide fragment NITEIADLTQK, and it is spy to retrieve the peptide fragment through BLAST
Different in nature peptide fragment, theoretical molecular is 1245.7Da；Synthesis peptide molecular weight is 1245.41Da, in the solution predominantly divalent charge,
Molecular weight is 623.3Da；In specific peptide fragment L amino acid sites label isotopes13C6 15N, label isotope molecular weight is 7Da,
Isotope marks peptide fragment molecular weight is 1252.6Da, in the solution predominantly divalent charge, and molecular weight is 626.8Da；Instrument
The triple level Four bar mass spectrographs of AB5500 identify that specific peptide fragment ion pair is 623.3/1018.6,623.3/675.1,623.3/
788.6；Isotope marks peptide fragment ion pair is 626.8/1025.6,626.8/682.2,626.8/795.5, wherein 623.3/
1018.6 is absolute quantitation ion pair with 626.8/1025.6.
3. the method that peptide fragment isotope dilution mass spectrometry according to claim 1 quantitatively recombinates Troponin I content, it is special
Levy and be, in described step (4), sample analysis pre-treatment is concretely comprised the following steps：
I) it is denatured, 25 μ L protein liquids samples add the μ L of 1ng/mL isotope marks peptide 7.5, then add 217.5 μ L25mmol/L carbonic acid
Hydrogen ammonium is mixed, plus urea 96mg makes its final concentration of 6M, is mixed, and stands 5min；
II) reductive alkylation, plus 250 μ L25mmol/L ammonium hydrogen carbonate, plus the μ L of 100mM dithiothreitol (DTT)s 50,60 DEG C of water-baths
30min；Alkylation, plus the μ L of 550mM iodoacetamides 50；Room temperature lucifuge 20min；Plus 200mM DTT50 μ L, room temperature 20min；Plus
The μ L of 25mmol/L ammonium hydrogen carbonate 1000, make urea concentration be less than 1M；
III) digest, add pancreatin, pancreatin and sample quality ratio are 1: 10,37 DEG C of water-bath 4h, plus the μ L ends of 0.1% trifluoroacetic acid 30
Only react；
IV) purification of samples, plus 3cc/60 μm of 3.0mL methyl alcohol activation OASIS MAX solid-phase extraction columns, add after methyl alcohol flows to end
3.0mL deionizations balance pillar, plus enzymolysis product is in post, is successively washed with 2.0mL10% ammoniacal liquor, 2.0mL methyl alcohol, finally uses
2.0mL2% formic acid methyl alcohol is eluted；
V) concentrating sample, the concentrate eluant on Nitrogen evaporator, gas flow 5mL/min, temperature 45 C treats that organic solvent volatilizees
Afterwards, dissolved with 0.1mL0.1%FA, upper machine carries out liquid chromatography mass spectrometric detection, the μ L of sample size 5.
4. the method that peptide fragment isotope dilution mass spectrometry according to claim 1 quantitatively recombinates Troponin I content, it is special
Levy and be, in described step (5), the specific method of data analysis is：
CTnI specificity peptide fragment concentration, cTnI protein peptides appearance time 4.20min, with software Analyst1.2.1 analytical standards
Peptide/Isotopic Internal Standard peptide peak area simultaneously makes standard curve, weighted line regress equation (" 1/x " weighting)：Y=
0.208x-0.072 (r=0.9988), cTnI peptide fragment concentration is calculated according to standard curve；It is prepared by cTnI peptides standard curve：Standard
Peptide amino acid isotope dilution mass spectrometry is traced to the source SI units, is used mass spectrum level water to prepare various concentrations 1ng/mL, 2.5ng/
ML, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, are separately added into the μ L of 1ng/mL isotope marks peptide 7.5 mixings, before detection
Processing procedure is identical with step (1)-step (4).
5. the method that peptide fragment isotope dilution mass spectrometry according to claim 1 quantitatively recombinates Troponin I content, it is special
Levy and be, in described step (6), content is that the 62.1ng/mL aqueous solution suitable peptide concentration 3.22ng/mL, 621ng/mL are water-soluble
Liquid phase works as peptide concentration 32.2ng/mL；CTnI protein recoveries low, high are respectively 94.65% and 103.15%；CTnI is low, sample high
Product betweenrun precision is respectively 7.04%, 5.69%, and withinrun precision is respectively 6.15%, 4.76%；The rate of recovery and precision
Meet regulation requirement.
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