CN106770821B - Method for quantifying content of recombinant troponin I by peptide isotope dilution mass spectrometry - Google Patents
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Abstract
A method for quantifying the content of recombinant troponin I by a peptide isotope dilution mass spectrometry method, which relates to a method for accurately quantifying the content of recombinant troponin I; the method comprises the following steps: designing a characteristic peptide segment; determining a characteristic peptide fragment mass spectrometry ion pair; study of LC-MRM conditions; sample analysis pretreatment: the method mainly comprises the following steps: denaturation of recombinant protein, reduction alkylation, enzymolysis, sample purification and sample concentration; and (3) data analysis: the concentration of the cTnI specific peptide fragment and the peak-off time of cTnI protein peptide are 4.20 min; recovery rate and precision: the recovery rate and precision meet the specified requirements, the conditions of the liquid phase mass spectrum of the recombinant cTnI specific peptide segment are researched and improved, the established method has good accuracy and high speed, and the peak-off time is 4.20 min.
Description
Technical Field
The invention relates to a method for accurately quantifying the content of recombinant troponin I, in particular to a method for quantifying the content of recombinant troponin I by a peptide isotope dilution mass spectrometry.
Background
Troponin (cTnI) is one of the most sensitive biological indicators for detecting myocardial injury. However, the cTnI detection still has some problems, mainly that the detection method is not standardized, certain differences exist among different methods, and the detection result often does not have space-time comparability.
Isotope Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has the advantages of high sensitivity, high accuracy and high specificity, and the standardization of the measurement result can be directly traced to the international basic unit system. The domestic scholars [1] establish the amino acid isotope dilution mass spectrometry method for quantitative recombination of cTnI, the method needs high-temperature hydrolysis of amino acid, and potential safety hazards exist.
The domestic research on the quantitative protein content by the peptide fragment isotope dilution mass spectrometry has few reports, is mainly complex with protein sample components, is easy to be influenced by other impurity ions in the ionization degree, and has more complex experimental conditions.
Reference documents:
[1] sunxueqing, an absolute quantitative method of important biomarkers, namely alpha fetoprotein, leptin and troponin I [ D ]. Beijing university of chemical industry, 2014.
[2]Kuhn E，Addona T，Keshishian H，et al.Developing multiplexed assaysfor troponin I and interleukin-33in plasma by peptide immunoaffinityenrichment and targeted mass spectrometry.Clin Chem[J].2009，55(6)：1108-17。
[3]Keshishian H，Addona T，Burgess M，et al..Quantification ofcardiovascular biomarkers in patient plasma by targeted mass spectrometry andstable isotope dilution.Mol Cell Proteomics[J].2009，8(10)：2339-49。
[4]Huillet C，Adrait A，Lebert D，et al.Accurate quantification ofcardiovascular biomarkers in serum using Protein Standard AbsoluteQuantification(PSAQTM)and selected reaction monitoring[J].Molecular&CellularProteomics Mcp，2012，11(2)：8681-8696。
[5]Zhao C，Trudeau B，Xie H，et al.Epitope mapping and targetedquantitation of the cardiac biomarker troponin by SID-MRM massspectrometry.Proteomics[J].2014，14(11)：1311-21。
Disclosure of Invention
The invention aims to provide a method for quantitatively determining the content of recombinant troponin I by a peptide fragment isotope dilution mass spectrometry, which is used for researching and improving the conditions of recombinant cTnI specific peptide fragment liquid phase mass spectrometry, and has the advantages of good accuracy, high speed and peak emergence time of 4.20 min.
In order to solve the problems existing in the background technology, the invention adopts the following technical scheme: a method for quantifying the content of recombinant troponin I by peptide fragment isotope dilution mass spectrometry comprises the following steps:
(1) designing a characteristic peptide fragment: searching a Pubmed protein database for the amino acid sequence of the human Troponin I, Access: NP _000354.4, then online analysis of mass-spec peptide fragments, website: http: expasy.org/peptide _ mass/, selecting a target peptide segment by combining the characteristics of a laboratory mass spectrometer, and performing blast homologous retrieval on the target peptide segment in a pubmed protein database to determine the target peptide segment as a specific peptide segment.
(2) Determination of ion pairs by characteristic peptide fragment mass spectrometry: and analyzing and researching parent ions and daughter ions of the specific peptide fragments by using skyline software, adjusting collision energy, and selecting three pairs of ions with strong signals for research, wherein the ions with the strongest signals are quantitative ion pairs, and the other ions are qualitative ion pairs.
(3) Study of LC-MRM conditions: ultra high pressure liquid chromatography (UPLC LC-30A) parameters liquid chromatograph: shimadzu liquid chromatography infusion pump and automatic sample injector; a chromatographic column: CSH 130C 18; column temperature 40 ℃, mobile phase a: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile solution, flow rate 0.3mL/min, mobile phase B2 min 5%, 8min 50%, 9min 90%, 14min 5%. Mass spectrum separation conditions, instrument AB 5500, ion spray voltage 5500V; the temperature is 550 ℃; the air curtain gas (CUR) is 25L/min, and the collision gas (CAD) is 8L/min; detecting in a positive ion mode; the scanning mode is Multiple Reaction Monitoring (MRM).
(4) Sample analysis pretreatment: the method mainly comprises the following steps: denaturation of recombinant protein, reduction alkylation, enzymolysis, sample purification and sample concentration.
(5) And (3) data analysis: the peak areas of the standard peptide (ion pair 623.3/1018.6)/isotope internal standard peptide (ion pair 626.8/1025.6) were analyzed by software Analystl.2.1 and a standard curve was generated. The cTnI peptide fragment concentration was calculated according to the standard curve. The concentration (ng/mL) of the cTnI of the recombinant protein is the concentration of the cTnI specific peptide fragment multiplied by 19.28; 19.28 dividing the molecular weight of cTnI (24016Da) by the molecular weight of the cTnI signature peptide (1245.41 Da).
(6) Recovery rate and precision: preparing aqueous solution with the cTnI content of 62.1ng/mL and 621ng/mL, and taking the ratio of a measured value to a theoretical value as the method recovery rate according to sample analysis pretreatment and measurement. Meanwhile, the low and high concentration protein samples are continuously measured for 5 times within 1d and for 5 days, respectively, and the intra-batch and inter-batch precision is calculated.
As a further improvement of the present invention; the specific operation method of the step (1) comprises the following steps: according to the amino acid sequence of the humanized cTnI protein, isotope dilution mass spectrometry is designed by Peptide mass software to detect a cTnI specific Peptide segment NITEIADLTQK, the Peptide segment is searched by BLAST to be a specific Peptide segment, and the theoretical molecular weight is 1245.7 Da; the molecular weight of the synthesized peptide is 1245.41Da, the synthesized peptide mainly has divalent charges in solution, and the molecular weight is 623.3 Da; labeling isotope (L) at amino acid position of specific peptide segment13C6 15N), the molecular weight of the labeled isotope is 7Da, the molecular weight of the isotope labeled peptide fragment is 1252.6Da, the isotope labeled peptide fragment mainly has divalent charges in solution, and the molecular weight is 626.8 Da; the specific peptide fragment ion pairs identified by an AB 5500 triple quadrupole mass spectrometer are 623.3/1018.6, 623.3/675.1 and 623.3/788.6; the ion pairs of the isotopically labeled peptide fragments are 626.8/1025.6, 626.8/682.1 and 626.8/795.5, wherein 623.3/1018.6 and 626.8/1025.6 are absolute quantitative ion pairs.
As a further improvement of the present invention; in the step (4), the sample analysis pretreatment comprises the following specific steps:
I) denaturing, adding 7.5 mul of 1ng/mL isotope labeled peptide into 25 mul of protein solution sample, adding 217.5 mul of 25mmol/L ammonium bicarbonate, mixing, adding 96mg of urea to make the final concentration be 6M, mixing, and standing for 5 min;
II) reductive alkylation, adding 250 μ L of 25mmol/L ammonium bicarbonate and 50 μ L of 100mM Dithiothreitol (DTT), and water bath at 60 deg.C for 30 min. Alkylation, 50. mu.L of 550mM Iodoacetamide (IAA) was added. Protected from light at room temperature for 20 min. Add 200mM DTT 50. mu.L, room temperature 20 min. Adding 1000 mu L of 25mmol/L ammonium bicarbonate to make the concentration of urea less than 1M;
III) carrying out enzymolysis, adding pancreatin, wherein the mass ratio of the pancreatin to the sample is 1: 10, carrying out water bath at 37 ℃ for 4h, and adding 30 mu L of 0.1% trifluoroacetic acid (TFA) to terminate the reaction;
IV) purifying the sample, adding 3.0mL of methanol to activate the OASIS MAX solid phase extraction column (3cc/60 μm), adding 3.0mL of deionization balance column after the methanol is drained, adding the enzymatic hydrolysate into the column, washing with 2.0mL of 10% ammonia water and 2.0mL of methanol in sequence, and finally eluting with 2.0mL of 2% formic acid methanol;
v) concentrating the sample, concentrating the eluent on a nitrogen blowing instrument, wherein the gas flow is 5mL/min, the temperature is 45 ℃, and after the organic solvent is volatilized, 0.1mL of 0.1% FA is used for dissolving. And (4) performing liquid mass spectrometry detection on the sample by using a machine, wherein the sample volume is 5 mu L.
As a further improvement of the present invention; in the step (5), the specific method for data analysis is as follows:
cTnI-specific peptide fragment concentration, cTnI protein peptide peak time 4.20min, standard peptide (ion pair 623.3/1018.6)/isotope internal standard peptide (ion pair 626.8/1025.6) peak area were analyzed and standard curve was made with software analyst1.2.1, weight linear regression equation ("1/x" weighing): y is 0.208x-0.072(r is 0.9988), and the concentration of the cTnI peptide fragment is calculated according to a standard curve; preparation of a cTnI peptide standard curve: and (3) diluting mass spectrometry traceable SI units with amino acid isotopes for standard peptides, preparing the standard peptides into mass spectrometry grade water with different concentrations of 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL and 100ng/mL, respectively adding 7.5 mu L of isotope labeled peptides with 1ng/mL, and uniformly mixing, wherein the detection pretreatment process is the same as the step (4) in the step (1).
As a further improvement of the present invention; in the step (6), the content is 62.1ng/mL of aqueous solution equivalent peptide concentration is 3.22ng/mL, and 621ng/mL of aqueous solution equivalent peptide concentration is 32.2 ng/mL; the recovery rates of the low cTnI protein and the high cTnI protein are 94.65% and 103.15%, respectively. The intercity precision (CV) of the cTnI low and high samples was 7.04% and 5.69%, respectively, and the intracity precision (CV) was 6.15% and 4.76%, respectively. The recovery rate and the precision meet the specified requirements.
After the technical scheme is adopted, the invention has the following beneficial effects:
and (3) screening out a cTnI specific peptide fragment according to a Pubmed database, and establishing a method for quantitatively recombining cTnI by a peptide fragment isotope dilution mass spectrometry method. The method is obviously different from the method for quantitatively recombining troponin I by an amino acid isotope dilution mass spectrometry in the aspects of a standard substance, an isotope internal standard and the like. The detection result of the method can be traced to the SI. Lays a foundation for the research of the serum cTnI standard substance.
Drawings
FIG. 1 is a diagram showing the selection of a bioinformatic analysis-specific peptide fragment according to an embodiment of the present invention;
in http: performing cTnI pancreatin enzyme cutting site analysis on a database, wherein the figure 1 shows the univalent and bivalent prediction results of the cTnI pancreatin enzyme cutting site, the characteristics of a mass spectrometer are combined, and the box is the final selection result;
FIG. 2 is a second scan of ion mass spectra of specific peptide fragments according to an embodiment of the present invention;
FIG. 3 is a second scan of an ion mass spectrum of an isotopically labeled peptide fragment according to an embodiment of the present invention;
fig. 4 is a diagram of cTnI LC-MS/MS according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The specific implementation mode adopts the following technical scheme: a method for quantifying the content of recombinant troponin I by peptide fragment isotope dilution mass spectrometry comprises the following steps:
(1) designing a peptide fragment:
according to the amino acid sequence of the human cTnI protein, the Peptide fragment NITEIADLTQK specific to cTnI is determined by Peptide mass software design isotope dilution mass spectrometry, and is searched as the specific Peptide fragment by BLAST, and the theoretical molecular weight is 1245.7Da as shown in figure 1. The molecular weight of the synthesized peptide is 1245.41Da, the synthesized peptide is mainly divalent charge in solution, and the molecular weight is 623.3 Da. We label the isotope(s) at the L amino acid position of the specific peptide segment13C6 15N), standardThe molecular weight of the isotope is 7Da, the molecular weight of the isotope labeling peptide segment is 1252.6Da, the isotope labeling peptide segment is mainly divalent charge in solution, and the molecular weight is 626.8 Da. The specific embodiment of the invention is different from the foreign reports in marking peptide fragments, and is shown in table 1:
TABLE 1 comparison of cTnI specific peptides and isotopically labeled peptides
The specific peptide fragment ion pairs identified by an AB 5500 triple quadrupole mass spectrometer are 623.3/1018.6, 623.3/675.1 and 623.3/788.6. The ion pairs of the isotopically labeled peptide fragments are 626.8/1025.6, 626.8/682.1 and 626.8/795.5. The results are shown in FIGS. 2 and 3, wherein 623.3/1018.6 and 626.8/1025.6 are absolute quantitative ion pairs.
(2) Liquid phase separation conditions:
ultra high pressure liquid chromatography (UPLC LC-30A) parameters liquid chromatograph: shimadzu liquid chromatography infusion pump and autosampler (japan corporation); a chromatographic column: the company Water CSH 130C18(150 mm. times.2.1, 3.5 μm, Waters corporation, USA); column temperature 40 ℃, mobile phase a: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile solution, flow rate 0.3mL/min, mobile phase B: 2min 5%, 8min 50%, 9min 90%, 14min 5%.
(3) Mass spectrum separation conditions:
the instrument AB 5500, ion spray voltage 5500V; the temperature is 550 ℃; the air curtain gas (CUR) is 25L/min, and the collision gas (CAD) is 8L/min; detecting in a positive ion mode; the scanning mode is Multiple Reaction Monitoring (MRM). The specific parameters are shown in Table 2:
table 2 conditions for mass spectrometry optimization
(4) Sample analysis pretreatment:
the method mainly comprises the following steps: denaturation of recombinant protein, reduction alkylation, enzymolysis, sample purification and sample concentration.
a) Denaturing, adding 7.5 μ L of 1ng/mL isotope labeled peptide into 25 μ L of protein solution sample, adding 217.5 μ L25 mmol/L ammonium bicarbonate, mixing, adding 96mg urea to make the final concentration 6M, mixing, and standing for 5 min.
b) Reductive alkylation, adding 250 μ L of 25mmol/L ammonium bicarbonate, adding 50 μ L of 100mM Dithiothreitol (DTT), and water bath at 60 deg.C for 30 min. Alkylation, 50. mu.L of 550mM Iodoacetamide (IAA) was added. Protected from light at room temperature for 20 min. Add 200mM DTT 50. mu.L, room temperature 20 min. Adding 1000 μ L of 25mmol/L ammonium bicarbonate to make the urea concentration less than 1M.
c) And (3) performing enzymolysis, adding pancreatin, wherein the mass ratio of the pancreatin to the sample is 1: 10, performing water bath at 37 ℃ for 4h, and adding 30 mu L of 0.1% trifluoroacetic acid (TFA) to terminate the reaction.
d) The sample was purified by adding 3.0mL of methanol activated OASIS MAX solid phase extraction column (3cc/60 μm), adding 3.0mL of deionised equilibration column after the methanol had run off, adding the enzymatic hydrolysate to the column, washing with 2.0mL 10% ammonia followed by 2.0mL methanol, and finally eluting with 2.0mL 2% formic acid methanol.
e) Concentrating the sample, and concentrating the eluate on a nitrogen blowing instrument at a gas flow rate of 5mL/min and a temperature of 45 deg.C, and dissolving with 0.1mL of 0.1% FA after the organic solvent is volatilized. And (4) performing liquid mass spectrometry detection on the sample by using a machine, wherein the sample volume is 5 mu L.
(5) Preparation of a cTnI peptide standard curve: standard peptides were diluted with amino acid isotopes mass spectrometry traceable SI units. The mixture was mixed with mass-spectrum-grade water to prepare 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL and 100ng/mL of different concentrations, and 7.5. mu.L of the peptide labeled with 1ng/mL isotope was added to the mixture, respectively, and mixed well. The detection pretreatment process is the same as the step 1-4.
(6) And (3) data analysis:
a) the concentration of cTnI specific peptide fragment and the peak time of cTnI protein peptide were 4.20min, as shown in FIG. 4. The peak areas of the standard peptide (ion pair 623.3/1018.6)/isotope internal standard peptide (ion pair 626.8/1025.6) were analyzed by software Analystl.2.1 and a standard curve was generated. Weighted linear regression equation ("1/x" weighting): y is 0.208x-0.072(r is 0.9988). The cTnI peptide fragment concentration was calculated according to the standard curve.
b) The concentration of the recombinant protein cTnI (ng/mL) is equal to the concentration of the cTnI-specific peptide fragment multiplied by 19.28. Note: 19.28 dividing the molecular weight of cTnI (24016Da) by the molecular weight of the cTnI signature peptide (1245.41 Da).
(7) Recovery and precision
An aqueous solution with the cTnI content of 62.1ng/mL (equivalent peptide concentration of 3.22ng/mL) and 621ng/mL (equivalent peptide concentration of 32.2ng/mL) is prepared, and the ratio of the measured value to the theoretical value is the method recovery rate according to the pretreatment and the measurement of the sample analysis. Meanwhile, the low and high concentration protein samples are continuously measured for 5 times within 1d, and continuously measured for 5d respectively, and the intra-batch and inter-batch precision is calculated. The recovery rates of the low cTnI protein and the high cTnI protein are 94.65% and 103.15%, respectively. The intercity precision (CV) of the cTnI low and high samples was 7.04% and 5.69%, respectively, and the intracity precision (CV) was 6.15% and 4.76%, respectively. The recovery rate and the precision meet the specified requirements.
The difference between the present embodiment and the prior art is as follows:
1. the selected peptide fragment is representative and is a cTnI-specific peptide. The peptide fragment of interest NITEIADLTQK is finally determined using bioinformatics techniques. Consistent with the reports of foreign scholars [2-4 ]]. When the UPLC-MS method is used for detecting the polypeptide, the response signal of the polypeptide is increased or inhibited relative to instruments and chemical reagents of reaction. Kuhn E et al [2]And Keshishidian H [3 ]]The isotope-specific peptide NITEIAD [ (A), (B), (C) and (C) is synthesized13C6)，L]TQK, 6Da more than standard peptide. Huillet C4]An isotope-specific peptide NITEIADLTQ [ ()13C6 15N2)K]8Da more than the standard peptide. Combines the characteristics of amino acid to synthesize isotope-specific peptide NITEIAD [ (A), (B), (C) and (C)13C6 15N)L]TQK, 7Da more than the standard peptide NITEIADLTQK (see Table 1). The physical and chemical properties of the isotope-specific peptide are completely consistent with those of target polypeptide, the expressed chromatographic mass spectrum behavior is also consistent, and the fluctuation of a detection response value caused by a matrix effect can be corrected.
And 2, an LC-MS/MS result shows that the detection system is AB Triple quad 5500LC/MS/MS, the system has good resolution and high sensitivity, and the mass spectrum optimization conditions are shown in Table 2. The detection conditions of Kuhn E by using an AB 4000Q TRAP LC/MS/MS detection system are basically consistent with those of the specific embodiment, Huillet C by using an API5500Q-TRAP detection system, and the literature does not provide mass spectrum optimization conditions. Kuhn E reports the strongest signal of ion pair 623.3/1018.5, peaking at 20 min. Huillet C reports that the signal of the ion pair 623.3/675.4 is strongest, 623.3/1018.5 times later, and the peak time is 36.2min, and the signal of the ion pair 623.3/1018.6 of the embodiment is strongest, and the peak time is 4.20 min. Zhao C, et al [5] establish another specific peptide fragment solution standard curve of cTnI, the lower limit of the standard curve is 4ng/mL, and the lower limit of the peptide solution standard curve established by the embodiment is 1 ng/mL.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (1)
1. The method for quantifying the content of the recombinant troponin I by the peptide isotope dilution mass spectrometry is characterized by comprising the following steps of:
(1) designing a specific peptide fragment: searching a Pubmed protein database for the amino acid sequence of the human Troponin I, Access: NP _000354.4, then online analysis of mass-spec peptide fragments, website: http: the method comprises the following steps of// web, expasy, org/peptide _ mass/, selecting a target peptide segment by combining the characteristics of a laboratory mass spectrometer, and then performing blast homologous retrieval on the target peptide segment in a pubmed protein database to determine the target peptide segment as a specific peptide segment;
(2) determination of ion pairs for mass spectrometry of specific peptide fragments: analyzing and researching parent ions and daughter ions of specific peptide fragments by using skyline software, adjusting collision energy, and selecting three pairs of ions with strong signals for research, wherein the ions with the strongest signals are quantitative ion pairs, and the other ions are qualitative ion pairs;
(3) study of LC-MRM conditions: ultra-high pressure liquid chromatography parameter liquid chromatograph: shimadzu liquid chromatography infusion pump and automatic sample injector; a chromatographic column: CSH 130C 18; column temperature 40 ℃, mobile phase a: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile solution, the flow rate is 0.3mL/min, the mobile phase B is 5% in 2min, 50% in 8min, 90% in 9min and 5% in 14 min; mass spectrum separation conditions, instrument AB 5500, ion spray voltage 5500V; the temperature is 550 ℃; the gas curtain gas is 25L/min, and the collision gas is 8L/min; detecting in a positive ion mode; the scanning mode is multi-reaction monitoring;
(4) sample analysis pretreatment: the method mainly comprises the following steps: denaturation of recombinant protein, reduction alkylation, enzymolysis, sample purification and sample concentration;
(5) and (3) data analysis: analyzing the peak area of the standard peptide/isotope internal standard peptide by using software Analyst1.2.1, making a standard curve, and calculating the concentration of the cTnI peptide fragment according to the standard curve; the concentration ng/mL of the cTnI of the recombinant protein is the concentration of the cTnI specific peptide fragment multiplied by 19.28; 19.28 dividing the cTnI molecular weight 24016Da by the cTnI characteristic peptide molecular weight 1245.41 Da;
(6) recovery rate and precision: preparing aqueous solution with the cTnI content of 62.1ng/mL and 621ng/mL, and taking the ratio of a measured value to a theoretical value as the method recovery rate according to sample analysis pretreatment and determination; meanwhile, the low and high concentration protein samples are respectively measured for 5 times within 1d, and for 5 days, and the intra-batch and inter-batch precision is calculated;
the specific operation method of the step (1) comprises the following steps: according to the amino acid sequence of the humanized cTnI protein, isotope dilution mass spectrometry is designed by Peptide mass software to detect a cTnI specific Peptide segment NITEIADLTQK, the Peptide segment is searched by BLAST to be a specific Peptide segment, and the theoretical molecular weight is 1245.7 Da; the molecular weight of the synthesized peptide is 1245.41Da, the synthesized peptide mainly has divalent charges in solution, and the molecular weight is 623.3 Da; marking isotope at L amino acid site of specific peptide segment13C6 15N, the molecular weight of the labeled isotope is 7Da, the molecular weight of the isotope labeled peptide fragment is 1252.6Da, the isotope labeled peptide fragment mainly has divalent charges in solution, and the molecular weight is 626.8 Da; the specific peptide fragment ion pairs identified by an AB 5500 triple quadrupole mass spectrometer are 623.3/1018.6, 623.3/675.1 and 623.3/788.6;the ion pairs of the isotope labeled peptide fragments are 626.8/1025.6, 626.8/682.1 and 626.8/795.5, wherein 623.3/1018.6 and 626.8/1025.6 are quantitative ion pairs;
in the step (4), the sample analysis pretreatment comprises the following specific steps:
I) denaturing, adding 7.5 mul of 1ng/mL isotope labeled peptide into 25 mul of protein solution sample, adding 217.5 mul of 25mmol/L ammonium bicarbonate, mixing, adding 96mg of urea to make the final concentration be 6M, mixing, and standing for 5 min;
II) reduction alkylation, adding 250 mu L of 25mmol/L ammonium bicarbonate and 50 mu L of 100mM dithiothreitol, and carrying out water bath at 60 ℃ for 30 min; alkylating, adding 550mM iodoacetamide 50 μ L; keeping away from light for 20min at room temperature; adding 200mM DTT 50 μ L, and standing at room temperature for 20 min; adding 1000 mu L of 25mmol/L ammonium bicarbonate to make the concentration of urea less than 1M;
III) carrying out enzymolysis, adding pancreatin, wherein the mass ratio of the pancreatin to the sample is 1: 10, carrying out water bath at 37 ℃ for 4h, and adding 30 mu L of 0.1% trifluoroacetic acid to terminate the reaction;
IV) purifying the sample, adding 3.0mL of methanol to activate the OASIS MAX solid phase extraction column for 3cc/60 mu m, adding 3.0mL of deionized water to balance the column after the methanol is completely drained, adding an enzymatic hydrolysate into the column, washing with 2.0mL of 10% ammonia water and 2.0mL of methanol in sequence, and finally eluting with 2.0mL of 2% formic acid methanol;
v) concentrating the sample, concentrating the eluent on a nitrogen blowing instrument, dissolving the eluent by 0.1mL of 0.1% FA after the organic solvent is volatilized at the temperature of 45 ℃ at the gas flow of 5mL/min, and performing liquid phase mass spectrometry on the sample, wherein the sample injection amount is 5 mu L;
in the step (5), the specific method for data analysis is as follows:
the concentration of cTnI specific peptide fragment, the peak-off time of cTnI protein peptide is 4.20min, the software analysis 1.2.1 is used for analyzing the peak area of standard peptide/isotope internal standard peptide and making a standard curve, and the weight linear regression equation is '1/x' weighing: y is 0.208x-0.072r is 0.9988, and the concentration of cTnI peptide fragment is calculated according to the standard curve; preparation of a cTnI peptide standard curve: diluting mass spectrometry traceable SI units by using amino acid isotopes for standard peptides, preparing the standard peptides into mass spectrometry grade water with different concentrations of 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL and 100ng/mL, respectively adding 7.5 mu L of isotope labeled peptides with 1ng/mL, and uniformly mixing, wherein the detection pretreatment process is the same as the steps (1) to (4);
in the step (6), the content is 62.1ng/mL of aqueous solution equivalent peptide concentration is 3.22ng/mL, and 621ng/mL of aqueous solution equivalent peptide concentration is 32.2 ng/mL; the recovery rates of the low cTnI protein and the high cTnI protein are 94.65 percent and 103.15 percent respectively; the inter-batch precision and the intra-batch precision of the cTnI low sample and the cTnI high sample are respectively 7.04 percent and 5.69 percent, and the intra-batch precision is respectively 6.15 percent and 4.76 percent; the recovery rate and the precision meet the specified requirements.
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| CN108957008B (en) * | 2018-08-07 | 2022-04-05 | 余鹏 | Characteristic peptide segment for detecting rat CYP2E1 enzyme, screening method and application thereof |
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| CN112342271A (en) * | 2020-10-20 | 2021-02-09 | 南通大学附属医院 | Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry |
| CN113281442A (en) * | 2021-05-27 | 2021-08-20 | 杭州广科安德生物科技有限公司 | Method for valuing standard substance rich in leucine alpha-2 glycoprotein 1 |
| CN113219117A (en) * | 2021-05-27 | 2021-08-06 | 杭州广科安德生物科技有限公司 | Mass spectrometry method of TIMP1 protein standard substance |
| CN114137124B (en) * | 2021-12-01 | 2024-04-12 | 北京中医药大学 | Method for carrying out rapid peptide map analysis on protein |
| CN114705849A (en) * | 2022-03-03 | 2022-07-05 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Mass spectrum method for absolute quantification of cystatin C in serum based on SILAC |
| CN115963194B (en) * | 2022-10-26 | 2024-09-03 | 中国计量科学研究院 | Insulin quantification method based on characteristic peptide isotope dilution mass spectrometry |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940986A (en) * | 2014-03-24 | 2014-07-23 | 安徽省煦棠医疗科技有限公司 | Preparation of troponin I specific locus antibody and detection kit thereof |
| CN105137071A (en) * | 2015-07-29 | 2015-12-09 | 安徽省煦棠医疗科技有限公司 | Cardiac troponin I homogenous enzyme immunoassay kit |
| CN205003164U (en) * | 2015-07-30 | 2016-01-27 | 广州天宝颂原生物科技开发有限公司 | Troponin I, brain sodium peptide, D - dimer ration joint inspection test paper strip |
| CN105759050A (en) * | 2016-01-29 | 2016-07-13 | 苏州联辰生物技术有限公司 | Immunofluorescence kit for quantitatively detecting content of troponin I and preparation method |
-
2016
- 2016-12-13 CN CN201611144062.5A patent/CN106770821B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940986A (en) * | 2014-03-24 | 2014-07-23 | 安徽省煦棠医疗科技有限公司 | Preparation of troponin I specific locus antibody and detection kit thereof |
| CN105137071A (en) * | 2015-07-29 | 2015-12-09 | 安徽省煦棠医疗科技有限公司 | Cardiac troponin I homogenous enzyme immunoassay kit |
| CN205003164U (en) * | 2015-07-30 | 2016-01-27 | 广州天宝颂原生物科技开发有限公司 | Troponin I, brain sodium peptide, D - dimer ration joint inspection test paper strip |
| CN105759050A (en) * | 2016-01-29 | 2016-07-13 | 苏州联辰生物技术有限公司 | Immunofluorescence kit for quantitatively detecting content of troponin I and preparation method |
Non-Patent Citations (4)
| Title |
|---|
| Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry;Eric Kuhn et al;《Clinical Chemistry》;20091231;第55卷(第6期);第1108-1117页 * |
| Epitope mapping and targeted quantitation of the cardiac biomarker troponin by SID-MRM mass spectrometry;Cheng Zhao et al;《Proteomics》;20141231;第14卷;第1311-1321页 * |
| Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution;Hasmik Keshishian et al;《Molecular & Cellular Proteomic》;20090713;第8卷(第10期);第2339-2349页 * |
| 重要生物标志物—甲胎蛋白、瘦素及肌钙蛋白I的绝对定量方法;孙雪晴;《中国优秀硕士学位论文全文数据库（工程科技Ⅰ辑）》;20150615(第6期);第B016-59页 * |
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