CN117491645A - Method for determining interleukin-6 based on peptide isotope dilution mass spectrum - Google Patents
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Abstract
The invention discloses a method for setting value of interleukin-6 based on peptide fragment isotope dilution mass spectrum, which comprises the following steps: 1) Selection of characteristic peptide fragments: selecting three peptide fragments of EK, ER and VK as characteristic peptide fragments, and the sequences of the three peptide fragments are shown in SEQ ID NO: 1-3; 2) Chemically synthesizing three characteristic peptide fragments and corresponding isotope labeled peptide fragments; 3) Respectively measuring the purity of three characteristic peptide fragments by adopting an isotope dilution mass spectrometry of amino acid; 4) Carrying out enzymolysis on IL-6 by trypsin; 5) Accurately quantifying three characteristic peptide fragments in the enzymolysis liquid by adopting an isotope dilution mass spectrometry of the peptide fragments; 6) High performance liquid chromatography-isotope dilution mass spectrometry analysis of the IL-6 enzymolysis peptide fragment; 7) Calculation of IL-6 content. The invention obtains three characteristic peptide fragments of IL-6, which are all specific peptide fragments of IL-6, are not easy to be influenced by interference components, and improve the accuracy of quantitative results; compared with the isotope dilution mass spectrometry of amino acid, the method for determining the value has specificity.
Description
Technical Field
The invention relates to the technical field of protein standard substance fixed value, in particular to a method for fixing interleukin-6.
Background
Interleukin-6 (IL-6), abbreviated as Interleukin-6, is a multifunctional glycoprotein consisting of 212 amino acids, which is a cytokine. It has low content in healthy people, and when the organism is infected by bacteria, the content is positively related to the degree of bacterial infection. IL-6 plays a variety of roles in body pathology and physiological activity, such as acute inflammatory response, autoimmune disease, coronary heart disease, tumor formation, sepsis, etc. Continuous monitoring of IL-6 levels in intensive care patients can effectively assess the severity of systemic inflammatory response syndrome, sepsis and the prognosis of septic shock. In addition, IL-6 can also be used as an early warning indicator of sepsis.
The study found that inflammatory cytokines were significantly elevated in serum of 2019-nCoV-infected patients, and thus 2019-nCoV infection was considered to be intimately involved in "cytokine storm". The expression level of IL-6 in patient serum contributes to the clinical typing of novel coronavirus infections and predicts disease severity and patient prognosis. The novel diagnosis and treatment method for coronavirus infection takes the progressive rise of peripheral blood inflammatory factor IL-6 as a novel clinical early warning index of severe and critical coronavirus infection. The determination of IL-6 content in serum has important significance for diagnosis and prognosis of various diseases.
At present, no IL-6 standard substance exists in China, and recombinant human interleukin 6 (rhIL-6) activity determination international standard substance (Code 89/548) developed by the institute of biological standards and verification (NIBSC) in the United kingdom is 1.0X10 5 IU/branch, 100,000IU/1.0ug is active standard substance. However, the existing standard substance cannot realize absolute quantification of protein content. Therefore, standard substances with accurate development value and traceability have urgent demands.
Isotope dilution mass spectrometry of amino acids is a common method for determining protein content, but the method cannot distinguish amino acids from proteins or impurities, and the method has high requirements on the purity of the proteins, and the fixed value result of the method may be higher.
Therefore, developing a method for realizing absolute quantification of interleukin-6 and traceability of measurement results to SI units becomes one of the technical problems to be solved in the art.
Disclosure of Invention
The invention aims to provide an interleukin-6 quantitative method based on peptide isotope dilution mass spectrometry. The method adopts isotope dilution mass spectrometry of peptide fragments to carry out fixed value, the selected peptide fragments are specific peptide fragments of IL-6, the specificity is good, the accuracy is high, and the measurement result can trace to SI unit kg.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for quantifying interleukin-6 based on peptide fragment isotope dilution mass spectrometry, comprising the steps of:
(1) Selection of characteristic peptide fragments: : carrying out enzymolysis on the interleukin-6 solution by trypsin, analyzing the enzymolysis solution by time-of-flight mass spectrometry and high-resolution mass spectrometry, and screening three characteristic peptide fragments of the interleukin-6: EALAENNLNLPK (EK) EFLQSLR (ER), VLIQFLQK (VK), SEQ ID NO: 1-3;
(2) Synthesis of characteristic peptide fragments: synthesizing characteristic peptide segments EK, ER, VK and three isotope labeled peptide segments, wherein the sequences of the three isotope labeled peptide segments are respectively EAL # 13 C 6 , 15 N)AENNLNLPK、EFL( 13 C 6 , 15 N)QSSLR、VL( 13 C 6 , 15 N) IQFLQK, three synthesized characteristic peptide fragments are used for quantification of interleukin-6;
(3) Accurate quantification of the purity of the characteristic peptide fragment: isotopically labeled with leucine, valine, proline and phenylalanine standard substances 13 C 9 Phenylalanine (Phe), 13 C 5 Valine (valine), 13 C 5 Proline, D 10 -leucine is an internal standard, and amino acid isotope dilution mass spectrometry is adopted to quantify three synthesized characteristic peptide fragments respectively;
(4) Subjecting interleukin-6 to trypsin enzymatic hydrolysis: optimizing the mass ratio of the sample to the enzyme, and plotting the mass spectrum peak area ratio of the enzyme-cleaved peptide fragment to the labeled peptide fragment, wherein the enzymolysis time with the highest ratio is used as the optimal enzymolysis time of the IL-6, and the mass ratio of the sample to the enzyme with the highest ratio is used as the mass ratio of the sample to the enzyme in the pretreatment process of the IL-6; the method comprises the steps of carrying out a first treatment on the surface of the
(5) Accurately quantifying three characteristic peptide fragments in the enzymolysis liquid by adopting an isotope dilution mass spectrometry of the peptide fragments;
(6) Optimization of three characteristic peptide fragment liquid phase conditions and mass spectrum conditions: determining characteristic peptide fragments and parent ions and child ions of the isotope labeled peptide fragments, optimizing mass spectrum parameters, carrying out mass spectrum analysis on the characteristic peptide fragments by adopting high performance liquid chromatography tandem mass spectrometry, and adopting a multi-reaction monitoring (MRM) mode in an acquisition mode; the method comprises the steps of carrying out a first treatment on the surface of the
(7) And respectively calculating the contents of three characteristic peptide fragments in the enzymolysis liquid, and calculating the concentration of IL-6 by using the contents of the characteristic peptide fragments as a constant value result of the interleukin-6 standard substance.
Wherein, the amino acid sequence of the interleukin-6 is shown as SEQ ID NO: 4.
Wherein in the step (2), three characteristic peptide fragments (EALAENNLNLPK (EK), EFLQSLR (ER), VLIQFLQK (VK)) and isotope labeled characteristic peptide fragments (EAL #) 13 C 6 , 15 N)AENNLNLPK、EFL( 13 C 6 , 15 N)QSSLR、VL( 13 C 6 , 15 N) IQFLQK); the three synthesized characteristic peptide fragments were used for quantification of interleukin-6.
In the step (3), the characteristic peptide fragment is accurately quantified, specifically:
1) The method comprises the steps of respectively adopting an isotope dilution mass spectrometry of amino acid to accurately fix EK, ER and VK, adopting an isotope dilution mass spectrometry of leucine and proline to accurately fix a peptide fragment EK, adopting an isotope dilution mass spectrometry of leucine and phenylalanine to accurately fix a peptide fragment ER, adopting an isotope dilution mass spectrometry of valine and phenylalanine to accurately fix VK, and respectively taking the average value of the two amino acid fixed value results as a fixed value result of a characteristic peptide fragment.
2) The exact concentration of the characteristic peptide fragment solution was calculated using the following formula.
Wherein C is AA : concentration of amino acid in the solution, μg/g;
p: amino acid standard purity,%;
m label (C) : adding the mass of the marked amino acid and ng into the peptide fragment;
R sample : amino acid chromatographic peak area ratio, unlabeled/labeled in sample;
I 1 : high target amino acid actual mass ratio, unlabeled/labeled;
I 2 : low target amino acid actual mass ratio, unlabeled/labeled;
R 1 : high standard amino acid peak area ratio, unlabeled/labeled;
R 2 : low standard amino acid peak area ratio, unlabeled/labeled;
m: EK. Quality of ER, VK solution, mg;
wherein C is i Represents the concentration of the characteristic peptide fragment solution calculated from the amino acid, μg/g; c (C) AA Represents the concentration of amino acid in the solution, μg/g; m is M i Representing the relative molecular mass of the peptide fragment of the corresponding characteristic, da; m is M AA Represents the relative molecular mass of each amino acid, da; n represents the number of corresponding amino acids in EK, ER, and VK.
Firstly, respectively accurately quantifying the concentrations of three characteristic peptide fragments by adopting an isotope dilution mass spectrometry of amino acid, and then accurately quantifying the purities of the synthesized three characteristic peptide fragments according to the dilution multiple of a sample;
the purity of the synthesized three characteristic peptide fragments was calculated using the following formula:
wherein P is i Represents the purity of EK, ER, VK,%; c (C) i Represents the measured concentrations of EK, ER, VK, μg/g; m is m 1 G represents the mass of the solution of the configured characteristic peptide fragment; m is m 2 Represents the mass of EK, ER, VK in solution, μg.
In the step (4), the enzymolysis time of IL-6 and the mass ratio of the sample to trypsin are optimized, the mass spectrum peak area ratio of the enzyme-cleaved peptide fragment to the internal standard peptide fragment is plotted, the enzymolysis time with the highest ratio is used as the optimal enzymolysis time of IL-6, and the mass ratio of the sample to the enzyme with the highest ratio is used as the mass ratio of the sample to the enzyme in the pretreatment process of IL-6. The enzymolysis time of the interleukin-6 is determined to be 12 hours, and the ratio of the sample to the trypsin is 30:1.
Wherein, the step (5) specifically comprises the following steps:
1) Preparing non-labeled characteristic peptide fragment mixed solution and isotope labeled peptide fragment mixed solution with corresponding concentrations according to the content of three characteristic peptide fragments after IL-6 enzyme digestion;
2) And accurately weighing the IL-6 solution with the volume to be measured, and adding and weighing the isotope labeled characteristic peptide fragment solution with the corresponding volume according to the mass of the three target peptide fragments obtained after the IL-6 enzyme digestion, so that the mass ratio of the characteristic peptide fragments to the labeled peptide fragments is 1:1.
3) And respectively preparing low-standard solution and high-standard solution according to the mass ratio of the non-marked characteristic peptide fragment to the marked characteristic peptide fragment of 0.9:1 and 1.1:1, and quantifying the concentration of the interleukin-6 solution.
Wherein, in the step (6), the chromatographic column used is ACQUITY UPLC Peptide BEH C, 2.1mm multiplied by 100mm and 1.7 μm; the mobile phase A is 0.1% formic acid water, the mobile phase B is 0.1% acetonitrile formate, the flow rate is 0.2ml/min, and the sample injection amount is2 mu L; flow gradient: (0-20) min, (10% -28%) B; (20-22) min; (28% -50%) B; (22-24) min, (50% -80%) B; (24.1-30) min,10% B; the ion source is ESI source, positive ion scanning mode, the acquisition mode adopts multi-reaction monitoring (MRM) mode, and the ion pair: EK (663.4/244.2), ER (490.3/462.3), VK (495.0/185.2), 13 C-EK(667.0/244.10)， 13 C-ER(493.7/590.2)， 13 C-VK (498.4/220.1); and the DP and CE values are optimized to maximize the mass spectrum signal value.
Wherein the step (7) is specifically to calculate the concentration of IL-6 by adopting the following formula,
wherein C is i : the concentration of EK, ER and VK in the enzymolysis liquid is mug/g;
p: purity of the characteristic peptide fragment,%;
m label (C) : adding the mass of the labeled peptide segment into the characteristic peptide segment, and ng;
R sample : the area ratio of the chromatographic peak of the characteristic peptide fragment in the sample is not marked/labeled;
I 1 : the actual mass ratio of the high-standard characteristic peptide fragment is not marked/marked;
I 2 : the actual mass ratio of the low-standard characteristic peptide fragment is not marked/marked;
R 1 : high standard characteristic peptide peak area ratio, non-mark/mark;
R 2 : low standard characteristic peptideSegment area ratio, unlabeled/labeled;
m: the mass of the IL-6 sample solution, mg;
wherein C is IL-6 : concentration of IL-6 in solution, μg/g; c (C) i : the concentrations of EK, ER, VK in the solution, μg/g; m is M IL-6 : the relative molecular mass of IL-6, da; n: the number of corresponding EK, ER, VK fragments in the IL-6 sequence; m is M i : EK. ER, VK, da.
Compared with the prior art, the invention has the outstanding effects that:
(1) The invention combines qualitative and quantitative methods, and improves the accuracy of the measurement result. The method of combining the time-of-flight mass spectrum, the high-resolution mass spectrum and the triple quadrupole tandem mass spectrometer is adopted, so that the qualitative method is combined with the quantitative method, the time-of-flight mass spectrum and the high-resolution mass spectrum select characteristic peptide fragments for the quantification of the triple quadrupole tandem mass spectrum, and the quantitative specific peptide fragments for triple quadrupole liquid chromatography-mass spectrometry are not easy to be influenced by interference components, thereby improving the accuracy of quantitative results.
(2) The method of the invention has the advantages that the IL-6 is fixed, the average value of the measurement results of 3 characteristic peptide fragments by isotope dilution mass spectrometry is selected, and the 3 characteristic peptide fragments are fixed by isotope labeled amino acids, so that the accuracy of the fixed result is improved.
(3) The invention is based on the peptide isotope dilution mass spectrometry method to determine the IL-6 mass concentration, as the standard value of the standard substance. Isotope dilution mass spectrometry is an internationally recognized potential benchmark measurement method, uses GBW 09236L-valine, GBW 09237L-leucine and GBW 09235L-phenylalanine purity national first-class standard substances as magnitude traceability reference standards, and uses measuring instruments such as weighing after verification/calibration, etc., so that magnitude traceability of standard substances to SI basic units of kilograms (kg) and moles (mol) is ensured.
The method for determining interleukin-6 based on peptide isotope dilution mass spectrometry according to the present invention will be further described with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a flow chart of a method for the quantification of interleukin-6 based on mass spectrometry of peptide fragment isotope dilution;
FIG. 2 is a TIC diagram of IL-6 enzymatic hydrolysis peptide sample Q-TOF;
FIG. 3 is a high resolution mass spectrum of IL-6 enzymatic hydrolysis peptide fragment;
FIG. 4 is a graph of different enzymatic hydrolysis time optimizations;
FIG. 5 is a graph of enzyme quantity optimization at different scales;
FIG. 6 is an MRM of IL-6 enzymatic peptide fragment and isotopic peptide fragment.
Detailed Description
The reagents and apparatus used in the following examples included:
reagent:
GBW 09237L-leucine purity standard: purity 99.8%, u=1.5% (k=2), national institute of metrology.
GBW09236 valine purity standard: purity 99.4%, u=0.6% (k=2), national institute of metrology.
GBW09235 phenylalanine purity standard: purity 99.8%, u=1.5% (k=2), national institute of metrology.
GBW (E) 100084 proline purity standard: purity 99.0%, u=1.5% (k=2), national institute of metrology.
13 C 9 Phenylalanine (Phe), 13 C 5 Proline, D 10 -a group consisting of leucine and leucine, 13 C 5 the valine isotopically labeled amino acid is of purity > 98% in Cambridge isotope laboratories, usa.
Acetonitrile, merck, germany, chromatographic grade.
EK. ER, VK, L-EK, L-ER, L-VK are synthesized by Nanjing Jinsri biotechnology Co.
Trifluoroacetic acid, sigma-Aldirich, usa, chromatographically pure;
formic acid, supelco, chromatograph;
trypsin, promega, usa;
dithiothreitol (DTT) Inalco, usa;
iodoacetamide (Iodoacetamide, IAM) inc.i.inalco;
instrument:
high performance liquid triple quadrupole mass spectrometer, AB SCIEX 5500,AB SCIEX Qtrap6500;
high performance liquid phase time of flight mass spectrometer, AB Q-TOF X500B;
an electronic balance: sidoris, XPE 56, MCA3.6P-2CCN-M;
high resolution mass spectrometer (nanoliter liquid phase): race moeid Orbitrap Exploris, 240;
ultra-high performance liquid chromatograph, waters BIO H-CLASS;
high performance liquid chromatograph, waters Alliance e2695.
Referring to FIG. 1, the interleukin-6 is determined based on peptide fragment isotope dilution mass spectrometry, and the specific steps are as follows:
1. and (3) selecting a characteristic peptide segment, carrying out enzymolysis on the IL-6 sample by trypsin, and carrying out time-of-flight mass spectrometry and high-resolution mass spectrometry on the sample after enzymolysis. The experimental operation steps are as follows:
(1) And (3) rinsing: mu.L of 50mM ammonium bicarbonate was added to 10k of the ultrafilter tube and rinsed.
(2) Solution replacement: centrifuge at 16000r for 10min. 100. Mu.L (1. Mu.g/uL) of sample was added to the ultrafiltration tube membrane.
(3) Denaturation: 100. Mu.L of 7M guanidine hydrochloride was added for denaturation.
(4) And (3) reduction: mu.L of 1M DTT was added thereto and the mixture was kept at 42℃for 1 hour.
(5) Alkylation: 10. Mu.L of 1M IAA was added at room temperature protected from light for 30min.
(6) Centrifuge, add 100 μl 50mM ammonium bicarbonate centrifuge, 16000g, centrifuge for 15min, until the ultrafiltration membrane solution is dry, repeat three times.
(7) Enzymolysis: the sample and trypsin are in a mass ratio of 30:1, and the temperature of the oven is kept constant for 12 hours at 37 ℃.
(8) The reaction was stopped by centrifugation in reverse for 1min with addition of FA, the final concentration of FA in the solution being 1%. Respectively analyzed by time-of-flight mass spectrometry and high-resolution mass spectrometry.
The time-of-flight tandem mass spectrometry system performs the following conditions:
chromatographic column: ACQUITY UPLC Peptide BEH C18 the color of the steel sheet is color,1.7 μm,2.1 mm. Times.100 mm, 1/kg, column temperature 30 ℃, mobile phase A:0.1% formic acid water, mobile phase B:0.1% acetonitrile formate and 2. Mu.L of sample were introduced.
Table 1 time-of-flight mass spectrometry mobile phase gradient table
| Time | Flow(mL/min) | A(％) | B(％) |
| 0 | 0.3 | 98.0 | 2.0 |
| 1.00 | 0.3 | 98.0 | 2.0 |
| 20.00 | 0.3 | 65.0 | 35.0 |
| 50.0 | 0.3 | 55.0 | 45.0 |
| 52.0 | 0.3 | 20.0 | 80.0 |
| 55.10 | 0.3 | 98.0 | 2.0 |
| 60.0 | 0.3 | 98.0 | 2.0 |
Mass spectrometry conditions: ion source: ESI source, positive ion scan, IDA mode, TOF MS scan range: (200-2000) Da, TOF MS/MS scanning range: (100-2000) Da, DP:100V, CE:15V.
The analytical software was BioPharmaView.
Orbitrap Exploris240 high-resolution mass spectrometer (nanoliter liquid phase) sample injection conditions are as follows:
liquid phase conditions: pre-column: acclaim PepMap TM 100 75μm×2cm，nanoViper 2Pk C18，3μm，Chromatographic column: acclaim PepMap TM RSLC 50μm×15cm，nanoViper C18，2μm，/>
Column temperature: room temperature, mobile phase a:0.1% formic acid water, mobile phase B:0.1% acetonitrile in water (80% acetonitrile +20% water); the sample injection amount was 1. Mu.L.
Table 2 high resolution mass spectrum mobile phase gradient table
| Time | Flow(nL/min) | A(％) | B(％) |
| 0 | 300 | 97.0 | 3.0 |
| 3 | 300 | 70.0 | 30.0 |
| 28.0 | 300 | 70.0 | 30.0 |
| 86.0 | 300 | 50.0 | 50.0 |
| 88.0 | 300 | 1.0 | 99.0 |
| 90.0 | 300 | 1.0 | 99.0 |
Mass spectrometry conditions: positive ion scan, scan range: (200-2000) Da, resolution: 15000, banding: 1-6.
The analytical software used was Thermo BioPharma finder5.1.
According to the results of the time-of-flight mass spectrum and the high-resolution mass spectrum, EK, ER and VK which can stably appear, have higher response intensity of the mass spectrum, have NO missed cleavage site and have proper sequence length are finally selected as characteristic peptide fragments, and the sequences of the EK, ER and VK are EALAENNLNLPK, EFLQSSLR, VLIQFLQK respectively, as shown in SEQ ID NO:1-3, the amino acid sequence of IL-6 is shown as SEQ ID NO: 4. The TIC diagram of the IL-6 enzymolysis peptide fragment sample under Q-TOF is shown in figure 2; the TIC diagram of the IL-6 enzymolysis peptide fragment sample under the high resolution mass spectrum is shown in figure 3.
2. Synthesis of characteristic peptide: the characteristic peptide segments EK, ER, VK and three isotope labeled peptide segments are synthesized, and the sequences of the three isotope labeled peptide segments are EA { L } 13 C 6 ， 15 N)}AENNLNLPK、EF{L( 13 C 6 ，15N)}QSSLR、V{L( 13 C 6 ， 15 N) } IQFLQK, the three synthesized characteristic peptide fragments were used for quantification of interleukin-6.
3. Accurate quantification of the purity of the characteristic peptide fragment: because the 3 characteristic peptide fragments for quantification are not standard substances with known purity, the peptide fragments are subjected to acid hydrolysis, and accurate value determination is carried out by using an isotope dilution mass spectrometry method so as to ensure traceability of a determination result, and the operation steps are as follows:
1) The method comprises the steps of respectively adopting an isotope dilution mass spectrometry of amino acid to accurately fix the EK, ER and VK solutions, adopting an isotope dilution mass spectrometry of leucine and proline to fix the EK solution of a peptide fragment, adopting an isotope dilution mass spectrometry of leucine and phenylalanine to fix the ER solution of the peptide fragment, adopting an isotope dilution mass spectrometry of valine and phenylalanine to fix the VK solution, and respectively taking the average value of the fixed value results of the two amino acids as the fixed value result of a characteristic peptide fragment.
2) Isotopic dilution mass spectrometry of amino acids: the EK, ER, VK were precisely weighed, dissolved in PBS buffer solution, and the mass of PBS buffer solution was precisely weighed. Placing the accurately weighed and configured EK, ER and VK solutions into ampoule bottles, adding a certain amount of marked amino acid, enabling the mass ratio of the theoretical content of the amino acid in each ampoule bottle to the added marked amino acid substance to be about 1, adding the same amount of leucine and proline marked standard solution into the EK solution, adding the same amount of leucine and phenylalanine marked standard solution into the ER solution, adding the same amount of valine and phenylalanine marked standard solution into the VK solution, centrifuging and concentrating to dryness, adding 800 mu L of 6mol/L hydrochloric acid solution, mixing uniformly, introducing nitrogen, deoxidizing and sealing, hydrolyzing for 36 hours in a 110 ℃, taking out nitrogen, blow-drying, adding 0.1% formic acid-water solution for re-dissolving, filtering with a 0.22 mu m filter membrane, and measuring.
3) Preparing low-standard and high-standard solutions according to the mass ratio of the non-marked amino acid to the marked amino acid of 0.9:1 and 1.1:1, centrifuging and concentrating the low-standard and high-standard solutions to dryness, and carrying out the same pretreatment on the sample.
4) And (3) performing liquid phase mass spectrometry on the isotope dilution of the amino acid, and optimizing mass spectrometry conditions.
Liquid phase conditions:
chromatographic column: ACQUITY UPLC Peptide BEH C18 Column,1.7 μm,2.1 mm. Times.100 mm, 1/kg, column temperature 30 ℃, mobile phase A:0.1% formic acid water, mobile phase B:0.1% acetonitrile formate, elution gradient: (0-10) min, (5-95)% B, (10-14) min,95% B, (14.10-16) min,5% B, flow rate 0.2mL/min.
Mass spectrometry conditions: the ion source is an ESI source, a positive ion scanning mode and a multi-reaction monitoring (MRM) mode is adopted in an acquisition mode. The sample injection conditions for isotope dilution mass spectrometry of amino acids are shown in table 3.
Table 3 amino acid isotope dilution mass spectrometry conditions
| ID | Q 1 | Q 3 | DP(V) | CE(V) |
| Phe | 166.1 | 120.1 | 50 | 19 |
| Pro | 116.1 | 70.0 | 50 | 23 |
| Val | 118.10 | 72.10 | 40 | 16 |
| Leu | 131.80 | 86.10 | 110. | 15 |
| 13 C 9 -Phe | 175.1 | 128.1 | 50 | 19 |
| 13 C 5 -Pro | 121.0 | 74.1 | 50 | 22 |
| 13 C 5 -Val | 123.10 | 76.10 | 40 | 15 |
| D 10 -Leu | 142.10 | 96.10 | 70 | 15 |
5) The concentration of the synthesized characteristic peptide fragment was calculated using the following formula
Wherein C is AA : concentration of amino acid in the solution, μg/g;
p: amino acid standard purity,%;
m label (C) : adding the mass of the marked amino acid and ng into the peptide fragment;
R sample : amino acid chromatographic peak area ratio, unlabeled/labeled in sample;
I 1 : high target amino acid actual mass ratio, unlabeled/labeled;
I 2 : low target amino acid actual mass ratio, unlabeled/labeled;
R 1 : high standard amino acid peak area ratio, unlabeled/labeled;
R 2 : low standard amino acid peak area ratio, unlabeled/labeled;
m: EK. The mass of ER, VK sample solution, mg;
wherein C is i Representing the calculated concentration of the characteristic peptide fragment of each amino acid, μg/g; c (C) AA Represents the concentration of amino acid in the solution, μg/g; m is M i Representing the relative molecular mass of the peptide fragment of the corresponding characteristic, da; m is M AA Represents the relative molecular mass of each amino acid, da; n represents the number of corresponding amino acids in EK, ER, and VK.
The purity of the synthesized three characteristic peptide fragments was calculated using the following formula:
wherein P is i Represents the purity of EK, ER, VK,%; c (C) i Represents the measured concentrations of EK, ER, VK, μg/g; m is m 1 G represents the mass of the solution of the configured characteristic peptide fragment; m is m 2 Represents the mass of EK, ER, VK in solution, μg.
TABLE 4 purity of peptide fragments
| Peptide fragment | EK | VK | ER |
| Concentration (%) | 92.15 | 93.89 | 93.52 |
4. The interleukin-6 standard is subjected to enzymolysis by trypsin, and enzymolysis conditions are optimized.
And accurately weighing an internal standard peptide fragment mixed solution, wherein the mass of each peptide fragment in the mixed solution is equivalent to that of each peptide fragment after enzymolysis of a sample. And mixing the IL-6 standard solution and the internal standard peptide fragment mixed solution according to the mass ratio of 1:1, and optimizing under the conditions of different enzymolysis time and different enzyme use amounts. The mass spectrum peak area ratio of the enzyme-cleaved peptide fragment and the internal standard peptide fragment is plotted, the enzymolysis time with the highest ratio is used as the optimal enzymolysis time of IL-6, and the mass ratio of the sample to the enzyme with the highest ratio is used as the mass ratio of the sample to the enzyme in the pretreatment process of IL-6. The enzymolysis time of the interleukin-6 is determined to be 12 hours, and the ratio of the sample to the trypsin is 30:1. The optimized diagram of different enzymolysis time is shown in figure 4, and the optimized diagram of different proportions of enzyme amounts is shown in figure 5.
5. The method adopts isotope dilution mass spectrometry of peptide fragments to accurately quantify three characteristic peptide fragments in enzymolysis liquid, and specifically comprises the following steps:
1) Preparing non-labeled characteristic peptide fragment mixed solution and isotope labeled peptide fragment mixed solution with corresponding concentrations according to the content of three characteristic peptide fragments after IL-6 enzyme digestion;
2) Accurately weighing the IL-6 solution with the volume to be measured, and adding and weighing the isotope internal standard solution with the corresponding volume according to the mass of three target peptide fragments obtained after the IL-6 enzyme digestion, so that the mass ratio of the characteristic peptide fragments to the internal standard peptide fragments is 1:1;
3) And preparing low-standard and high-standard solutions according to the mass ratio of the non-marked characteristic peptide fragment to the marked characteristic peptide fragment of 0.9:1 and 1.1:1, and quantifying the concentration of the interleukin-6 solution.
The operation steps are as follows:
accurately weighing a certain mass of IL-6 solution, adding a solution containing equivalent mixed labeled peptide fragments according to the mass of characteristic peptide fragments after enzyme digestion of a sample, adding 100 mu L of 50mM ammonium bicarbonate, 100 mu L of 7M guanidine hydrochloride and 4 mu L of 1M DTT, and standing at 42 ℃ for 1h. Subsequently, 10. Mu.L of 1M IAA was added at room temperature protected from light for 30min. And (3) adding 50mM ammonium bicarbonate solution for dilution, so that the concentration of guanidine hydrochloride is reduced to below 1mol/L, and carrying out enzymolysis on a sample and trypsin for 12 hours at 37 ℃ in a mass ratio of 30:1. The reaction was terminated by adding formic acid, and the final concentration of formic acid in the solution was 1%. Filtering membrane to be measured.
6. And optimizing the liquid phase condition and the mass spectrum condition of the three characteristic peptide fragments. And determining characteristic peptide fragment and isotopically labeled peptide fragment parent ions and daughter ions, and optimizing parameters such as DP, CE values and the like. The mass spectrum optimization parameters are shown in table 5.
TABLE 5 Mobile phase Mass Spectrometry conditions for peptide fragment isotope dilution Mass Spectrometry
And carrying out mass spectrometry on the characteristic peptide fragments by adopting high performance liquid chromatography tandem mass spectrometry, wherein a multi-reaction monitoring (MRM) mode is adopted for acquisition. Performing mass spectrometry on the characteristic peptide fragment by adopting high performance liquid chromatography tandem mass spectrometry, wherein the chromatographic column is ACQUITY UPLC Peptide BEH C, 2.1mm multiplied by 100mm and 1.7 mu m; the mobile phase A is 0.1% formic acid water, the mobile phase B is 0.1% acetonitrile formate, the flow rate is 0.2ml/min, and the sample injection amount is2 mu L; flow gradient: (0-20) min, (10% -28%) B; (20-22) min; (28% -50%) B; (22-24) min, (50% -80%) B; (24.1-30) min,10% B; the ion source is an ESI source, a positive ion scanning mode and a multi-reaction monitoring (MRM) mode is adopted in an acquisition mode.
The MRM diagram of the IL-6 enzymolysis peptide fragment and the isotope internal standard peptide fragment is shown in figure 6.
7. And respectively calculating the contents of three characteristic peptide fragments in the enzymolysis liquid, and calculating the concentration of IL-6 by using the contents of the characteristic peptide fragments as a constant value result of the interleukin-6 standard substance. IL-6 concentration was averaged over 3 characteristic peptide measurements.
The concentration of IL-6 was calculated using the following formula:
wherein C is i : the concentrations of EK, ER, VK in the solution, μg/g;
p: purity of characteristic peptide fragments EK, ER, VK,%;
m label (C) : adding the mass of the labeled peptide segment into the characteristic peptide segment, and ng;
R sample : the area ratio of the chromatographic peak of the characteristic peptide fragment in the sample is not marked/labeled;
I 1 : the actual mass ratio of the high-standard peptide fragment is not marked/marked;
I 2 : the actual mass ratio of the low-standard peptide fragment is not marked/marked;
R 1 : high peak area ratio of peptide fragment, unlabeled/labeled;
R 2 : low-standard peptide area ratio, unlabeled/labeled;
m: the mass of the IL-6 sample solution, mg;
wherein C is IL-6 : concentration of IL-6 in solution, μg/g; c (C) i : concentration of EK, ER, VK in solution,%; m is M IL-6 : the relative molecular mass of IL-6 is 20808.31Da; n: the number of corresponding EK, ER, VK fragments in the IL-6 sequence; m is M i : EK. ER, VK, da. The constant value result of interleukin-6 is the average value of the constant values of three characteristic peptide fragments of EK, ER and VK.
The IL-6 assay results are shown in Table 6 below.
TABLE 6IL-6 quantitative results of dilution mass spectrometry based on peptide fragment isotopes
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. A method for quantifying interleukin-6 based on peptide fragment isotope dilution mass spectrometry, comprising the steps of:
(1) Selection of characteristic peptide fragments: selecting EK, ER and VK as characteristic peptide fragments, wherein the sequences of the EK, ER and VK are EALAENNLNLPK, EFLQSSLR, VLIQFLQK respectively, and SEQ ID NO: 1-3;
(2) Synthesis of characteristic peptide: synthesizing characteristic peptide segments EK, ER, VK and three isotope labeled peptide segments, wherein the sequences of the three isotope labeled peptide segments are respectively EAL # 13 C 6 , 15 N)AENNLNLPK、EFL( 13 C 6 , 15 N)QSSLR、VL( 13 C 6 , 15 N)IQFLQK；
(3) Accurate quantification of the purity of the characteristic peptide fragment: isotope labeling with leucine, proline and phenylalanine standard substance 13 C 9 Phenylalanine (Phe), 13 C 5 Proline, D 10 -leucine is an internal standard, and the amino acid isotope dilution mass spectrometry is used for quantifying the three synthesized characteristic peptide fragments;
(4) Carrying out enzymolysis on interleukin-6 by trypsin;
(5) Accurately quantifying three characteristic peptide fragments in the enzymolysis liquid by adopting an isotope dilution mass spectrometry of the peptide fragments;
(6) Optimization of three characteristic peptide fragment liquid phase conditions and mass spectrum conditions: determining characteristic peptide fragment and isotopically labeled peptide fragment parent ions and child ions, and optimizing mass spectrum parameters;
(7) And respectively calculating the contents of three characteristic peptide fragments in the enzymolysis liquid, and calculating the concentration of IL-6 by using the contents of the characteristic peptide fragments as a constant value result of the interleukin-6 standard substance.
2. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: the amino acid sequence of the interleukin-6 is shown as SEQ ID NO: 4.
3. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: in the step (3), the peptide EK is quantified by dilution mass spectrometry of isotopes of leucine and proline, the peptide ER is quantified by leucine and phenylalanine, and the VK is quantified by valine and phenylalanine.
4. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: in the step (4), the enzymolysis time is 12 hours, and the ratio of the sample to the trypsin is 30:1.
5. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: the content of the synthesized characteristic peptide fragment is calculated by adopting the following formula,
wherein C is AA : concentration of amino acid in the solution, μg/g;
p: amino acid standard purity,%;
m label (C) : adding the mass of the marked amino acid and ng into the peptide fragment;
R sample : amino acid chromatographic peak area ratio in solution, unlabeled/labeled;
I 1 : high heightThe actual mass ratio of the target amino acid, unlabeled/labeled;
I 2 : low target amino acid actual mass ratio, unlabeled/labeled;
R 1 : high standard amino acid peak area ratio, unlabeled/labeled;
R 2 : low standard amino acid peak area ratio, unlabeled/labeled;
m: EK. The mass of ER, VK sample solution, mg;
wherein C is i Representing the calculated concentration of the characteristic peptide fragment of each amino acid, μg/g; c (C) AA Represents the concentration of amino acid in the solution, μg/g; m is M i Representing the relative molecular mass of the peptide fragment of the corresponding characteristic, da; m is M AA Represents the relative molecular mass of each amino acid, da; n represents the number of corresponding amino acids in EK, ER, and VK.
6. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: in the step (6), the chromatographic column used is ACQUITY UPLC Peptide BEH C, 2.1mm×100mm,1.7 μm; the mobile phase A is 0.1% formic acid water, the mobile phase B is 0.1% acetonitrile formate, the flow rate is 0.2ml/min, and the sample injection amount is2 mu L;
flow gradient: (0-20) min, (10% -28%) B; (20-22) min; (28% -50%) B; (22-24) min, (50% -80%) B; (24.1-30) min,10% B; ion pair: EK 663.4/244.2, ER 490.3/462.3, VK 495.0/185.2, 13 C-EK 667.0/244.10， 13 C-ER 493.7/590.2， 13 C-VK 498.4/220.1; and the DP and CE values are optimized.
7. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: the step (7) is specifically that,
1) Preparing characteristic peptide fragment and isotope labeled peptide fragment solutions with corresponding concentrations according to the quality of the three characteristic peptide fragments after enzyme digestion;
2) Preparing three characteristic peptide isotope internal standard solutions into mixed solutions with the mass ratio of peptide fragments in a sample solution being 1:1; the ratio of the peptide standard solution to the isotope internal standard is 0.9:1,1:1 and 1.1:1;
3) Weighing the IL-6 protein solution with the volume to be measured, adding and weighing the isotope internal standard mixed solution with the same volume, wherein the concentration of the characteristic peptide fragment of the IL-6 to be measured is in the middle position of the determined linear range;
4) IL-6 concentration was calculated using the following formula
Wherein C is i : the concentrations of EK, ER, VK in the solution, μg/g;
p: purity of the characteristic peptide fragment,%;
m label (C) : adding the mass of the labeled peptide segment into the characteristic peptide segment, and ng;
R sample : the area ratio of the chromatographic peak of the characteristic peptide fragment in the sample is not marked/labeled;
I 1 : the actual mass ratio of the high-standard peptide fragment is not marked/marked;
I 2 : the actual mass ratio of the low-standard peptide fragment is not marked/marked;
R 1 : high peak area ratio of peptide fragment, unlabeled/labeled;
R 2 : low-standard peptide area ratio, unlabeled/labeled;
m: the mass of the IL-6 sample solution, mg;
wherein C is IL-6 : concentration of IL-6 in solution, μg/g; c (C) i : the concentrations of EK, ER, VK in the solution, μg/g; m is M IL-6 : the relative molecular mass of IL-6, da;n: the number of corresponding EK, ER, VK fragments in the IL-6 sequence; m is M i : EK. ER, VK, da.
8. The method for the quantification of interleukin-6 based on the mass spectrum of peptide fragment isotope dilution according to claim 1, characterized in that: the method comprises the steps of (1) accurately quantifying the purity of the characteristic peptide fragments in the step (3), firstly, respectively quantifying the concentrations of the three characteristic peptide fragments by adopting an isotope dilution mass spectrometry of amino acid, and then, accurately quantifying the purity of the synthesized three characteristic peptide fragments according to the dilution multiple of a sample; the purity of the synthesized three characteristic peptide fragments was calculated using the following formula:
wherein P is i Represents the purity of EK, ER, VK,%; c (C) i Represents the measured concentrations of EK, ER, VK, μg/g; m is m 1 G represents the mass of the solution of the configured characteristic peptide fragment; m is m 2 Represents the mass of EK, ER, VK in solution, μg.
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