CN112098540A - Characteristic peptide fragment, detection method and kit - Google Patents

Abstract

The invention discloses a characteristic peptide segment, which at least comprises peptide segments with amino acid sequences of QGLLPVLESFK, LSPLGEEMR, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, VTTVASHTSDSDVPSGVTEVVVK and TLLSNLEEAK respectively. The invention also provides a detection method and a kit. The invention uses the isotope labeled peptide segment corresponding to the characteristic peptide segment as an internal standard substance to realize the simultaneous and quantitative detection of the concentration of apolipoprotein A, apolipoprotein E and apolipoprotein J in blood plasma or blood serum.

Description

Characteristic peptide fragment, detection method and kit

Technical Field

The invention relates to the technical field of protein detection. More particularly, the invention relates to characteristic peptide fragments related to quantitative detection of apolipoprotein A, apolipoprotein E and apolipoprotein J, a detection method and a kit.

Background

The protein portion of lipoproteins is called Apolipoprotein (Apo), which has important physiological functions in lipoprotein metabolism. The main functions of apolipoproteins are: maintaining the structure and lipid transportation of lipoprotein; regulating the activity of lipometabolism related enzyme; ③ as a ligand of a cell surface lipoprotein receptor and involved in the metabolism of lipoproteins. Apolipoproteins are generally classified into apolipoprotein a, apolipoprotein B, apolipoprotein C, apolipoprotein D, apolipoprotein E, apolipoprotein J, and the like, each of which is classified into several subtypes. Apolipoprotein A (Apolipoprotein A) is an important component constituting high-density lipoprotein in plasma, and has effects of removing tissue lipid and resisting atherosclerosis. Apolipoprotein A-II (ApoAII) is one of the most important components of the apolipoprotein A family. ApoAII hydrolyzes triglycerides in Chylomicrons (CM) and Very Low Density Lipoproteins (VLDL), and its serum concentration is inversely related to the risk of developing coronary heart disease. Apolipoprotein E (Apolipoprotein E) is a polymorphic protein, participates in the conversion and metabolic process of lipoprotein, the gene can regulate a plurality of biological functions, is related to the onset of a plurality of ophthalmic diseases, the research on ApoE and the gene polymorphism thereof is one of the hot spots of the current medical research, the internal relation between the ApoE and the gene polymorphism is discussed, and the Apolipoprotein E has important clinical application value for the prevention, diagnosis and treatment of eye diseases. ApoE is mainly present in CM, VLDL, Intermediate Density Lipoprotein (IDL) and part of High Density Lipoprotein (HDL), and the concentration of ApoE is positively correlated with the plasma triglyceride content. Apolipoprotein J (Apolipoprotein J, ApoJ) is a glycoprotein that binds HDL and VHDL, and is a dimeric protein with a molecular weight of about 70 kD. It is widely distributed in various tissues of human body, and is also involved in physiological processes such as complement function regulation, sperm maturation, membrane recycling and the like, and is related to the onset of certain diseases, besides being responsible for lipid transport.

The traditional quantitative research of macromolecular protein compounds is mainly carried out by adopting methods such as isoelectric focusing electrophoresis, two-dimensional gel electrophoresis, immunoblotting and the like, and has unique advantages for enzyme-linked immunosorbent assays (ELISA) and radioactive immunological labelling methods (RIA) of protein compounds with lower content. Among them, ELISA has become the most commonly used method for analyzing protein polypeptide compound biological samples because of its advantages such as higher sensitivity and suitability for batch measurement. However, the traditional immunization method has the defects of long development period, high cost, protein cross reaction and the like.

Disclosure of Invention

The invention aims to provide a characteristic peptide fragment, a detection method and a kit, which utilize an isotope labeled peptide fragment corresponding to the characteristic peptide fragment as an internal standard substance to realize simultaneous and quantitative detection of the concentrations of apolipoprotein A, apolipoprotein E and apolipoprotein J in plasma or serum.

To achieve these objects and other advantages in accordance with the present invention, there is provided according to one aspect of the present invention a characteristic peptide stretch comprising at least peptide stretches having amino acid sequences QGLLPVLESFK, LSPLGEEMR, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, VTTVASHTSDSDVPSGVTEVVVK and TLLSNLEEAK, respectively.

According to another aspect of the present invention, there is provided a detection method comprising using an isotopically labeled peptide fragment corresponding to the characteristic peptide fragment of claim 1 as an internal standard in the simultaneous detection of apolipoprotein A, apolipoprotein E and apolipoprotein J.

Further, the detection method comprises the following steps:

preparing an isotope labeled peptide fragment internal standard solution;

preparing a correction standard sample of the characteristic peptide fragment by using a substitute matrix;

treating a sample to be detected with a dithiothreitol solution, an iodoacetamide solution and trypsin in sequence, adding an isotope labeled peptide internal standard solution, loading onto a column, eluting, drying by nitrogen blowing or freezing, drying, and redissolving to obtain a sample solution to be detected;

adding an isotope labeled peptide fragment internal standard solution into the calibration standard sample, analyzing by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide fragment and the peak area of the corresponding isotope labeled peptide fragment, and establishing a standard curve taking the peak area ratio and the concentration as coordinates;

and analyzing the sample solution to be detected by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide segment and the peak area of the corresponding isotope labeled peptide segment, substituting the ratio of the peak areas into a standard curve, and calculating the concentrations of the apolipoprotein A, the apolipoprotein E and the apolipoprotein J.

Further, the detection method further includes:

preparing a quality control sample of the characteristic peptide segment by using a substitute matrix, adding an isotope labeled peptide segment internal standard solution into the quality control sample, analyzing by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide segment and the peak area of the corresponding isotope labeled peptide segment, substituting the ratio of the peak areas into a standard curve, and calculating the recovery rate value of the quality control sample.

Further, the detection method, the preparation method of the alternative matrix comprises the following steps:

taking bovine albumin, sequentially treating with dithiothreitol solution, iodoacetamide solution and trypsin, loading onto column, eluting, drying with nitrogen gas or freeze drying, and redissolving to obtain the substitute matrix.

Further, the detection method adopts a liquid chromatography-mass spectrometry combined instrument LC-MS/MS;

the mass spectrometry conditions include: the ion source is an electrospray ion source, the positive ion mode detection is adopted, and the scanning mode is multiple reaction monitoring.

Further, in the detection method, the parameters monitored by the multiple reactions comprise:

wherein VTTVASHTSDSDVPSGVTEVVVK, TLLSNLEEAK, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, QGLLPVLESFK and LSPLGEEMR respectively represent corresponding isotopically labeled peptide fragments of characteristic peptide fragment VTTVASHTSDSDVPSGVTEVVVK, TLLSNLEEAK, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, QGLLPVLESFK, LSPLGEEMR.

The chromatographic conditions include: the chromatographic column is Phenomenex Aeris WIDEPORE 3.6 μm XB-C18, 150 × 2.1 mm; the mobile phase is 0.1 percent of formic acid water solution-0.1 percent of formic acid acetonitrile, and gradient elution is carried out; the column temperature was 40 ℃; the amount of sample was 10. mu.L.

According to yet another aspect of the invention, there is provided a kit comprising:

an internal standard solution prepared from isotope-labeled peptide fragments corresponding to the characteristic peptide fragments in claim 1;

a calibration standard sample prepared from a substitute matrix and the characteristic peptide fragment;

dithiothreitol solution and iodoacetamide solution for treating the sample to be tested.

Further, the kit further comprises:

and the quality control sample is prepared from a substitute matrix and the characteristic peptide fragment, and at least comprises three different concentrations, namely high concentration, medium concentration and low concentration.

Further, the kit further comprises:

the buffer solution 1 is prepared from ammonium bicarbonate, urea and water, and the buffer solution 2 is prepared from ammonium bicarbonate and water.

The invention at least comprises the following beneficial effects:

the peptide segments with amino acid sequences of QGLLPVLESFK, LSPLGEEMR, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, VTTVASHTSDSDVPSGVTEVVVK and TLLSNLEEAK are used as characteristic peptide segments, the characteristic peptide segments are used for preparing a correction standard solution, isotope labeled peptide segments corresponding to the characteristic peptide segments are used as internal standard substances, and the concentration of apolipoprotein A, apolipoprotein E and apolipoprotein J in plasma or serum can be simultaneously and quantitatively detected by a liquid chromatography-mass spectrometry combined instrument.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a diagram showing the detection scheme of apolipoprotein A, apolipoprotein E, apolipoprotein J;

FIG. 2 is a two-stage full scan mass spectrum of characteristic peptide fragments and isotope internal standard [ M + H ] + of apolipoprotein A, apolipoprotein E, apolipoprotein J;

FIG. 3 is a typical extracted ion flow chromatogram of apolipoprotein A, apolipoprotein E, apolipoprotein J characteristic peptide fragment and an isotopic internal standard;

FIG. 4 is a typical standard graph of characteristic peptide fragments of apolipoprotein E and apolipoprotein J;

FIG. 5 is a typical standard curve diagram of a characteristic peptide fragment of apolipoprotein A;

FIG. 6 is a typical standard curve diagram of another characteristic peptide fragment of apolipoprotein A.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

In recent years, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology is continuously perfected and developed, and the LC-MS/MS can simultaneously obtain reliable qualitative and quantitative results, particularly, a mass spectrometry means represented by a Multiple Reaction Monitoring (MRM) technology can greatly reduce background noise and improve sensitivity and repeatability of an analysis method due to the fact that ions can be selected twice, and the LC-MS/MS technology becomes an important means for protein quantification. Compared with the traditional immunological method, the LC-MS/MS method has great advantages in the aspects of accuracy, selectivity, reproducibility, sensitivity, quantitative linear range and the like.

The invention relates to a protein quantitative analysis method based on a liquid chromatography tandem mass spectrometry technology, which comprises the steps of screening specific peptide fragments of proteins by using a shot-gun proteomics means, synthesizing peptide fragment standard substances and establishing a peptide fragment mass spectrometry quantitative analysis method. For the pretreatment process of the biological sample, the conditions of the steps of protein reduction, denaturation, pancreatin hydrolysis and the like are optimized to ensure the precision and reliability of the method, and finally, the analysis of the corresponding protein concentration level in the biological sample is realized through the quantification of the peptide fragment level. The kit takes a characteristic peptide standard substance added to a substitute matrix as a correction standard sample, takes a corresponding stable isotope labeled peptide as an internal standard, respectively treats the internal standard sample with Dithiothreitol (DTT) and Iodoacetamide (IAA), carries out enzymolysis through Trypsin (Trypsin), treats the internal standard sample with a Solid Phase Extraction (SPE) column, dries the internal standard sample with nitrogen or freezes to dry the internal standard sample, and redissolves the external standard sample to carry out liquid chromatography mass spectrometry detection. And recording peak areas of characteristic peptide fragments of the apolipoprotein A, the apolipoprotein E and the apolipoprotein J and an internal standard. And calculating the concentration of the characteristic peptide fragment by adopting an internal standard method according to the peak area/internal standard peak area ratio of the characteristic peptide fragment in the sample to be detected. The concentrations of apolipoprotein A, apolipoprotein E and apolipoprotein J were calculated according to the molar concentration.

The kit comprises the following components in parts by weight:

1. preparing characteristic peptide fragment correction standard samples and quality control samples of apolipoprotein A, apolipoprotein E and apolipoprotein J:

1) synthesizing a characteristic peptide fragment standard: the characteristic peptide fragment information of apolipoprotein A (ApoA1), apolipoprotein E (ApoE) and apolipoprotein J (ApoJ) is shown in a table 1 and a sequence table, and a characteristic peptide fragment standard is synthesized. The amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 in the sequence list are peptide segments QGLLPVLESFK, LSPLGEEMR, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, VTTVASHTSDSDVPSGVTEVVVK and TLLSNLEEAK, respectively.

TABLE 1 proteins and characteristic peptide fragments, stable isotope labeled peptide fragments

2) Preparing a characteristic peptide fragment stock solution: precisely weighing appropriate amount of each characteristic peptide fragment standard, dissolving with acetonitrile-water (v/v ═ 1:4) containing 0.1% Formic Acid (FA) to 1mg/mL each peptide fragment stock solution, and subpackaging and freezing at below-70 deg.C before use.

3) Preparation of alternative matrices for calibration standards and quality control samples: mu.L of 20mg/mL Bovine Serum Albumin (BSA) Buffer1 solution, 20. mu.L of 1M Dithiothreitol (DTT) aqueous solution, 880. mu.L of Buffer1, incubated at 60 ℃ for 1 h; recovering to room temperature, adding Iodoacetamide (IAA) 7.4mg, and keeping at 25 deg.C in dark for 40 min; adding 7mL of Buffer2, vortexing, adding 80 μ L of 1mg/mL Trypsin (Trypsin), and performing enzymolysis at 37 ℃ for 15 h; adding 100 mu L of FA to stop enzymolysis;

samples were loaded onto Solid Phase Extraction (SPE) cartridges (model: Oasis PRIME HLB Cartridge 1cc/30mg100 bx; or similar SPE Cartridge that had been activated and equilibrated) and washed 3 times with 1mL each time of 0.1% trifluoroacetic acid (TFA); the clean sample collection tube was replaced and eluted 2 times with 1mL each time of 0.1% TFA in acetonitrile-water (v/v ═ 4: 1); drying the eluent at 45 ℃ with nitrogen or freezing and drying; the blow-dried or freeze-dried sample can be stored in a refrigerator below 70 ℃ for a long time; 2.67 mL0.1% FA as surrogate matrix for calibration standards and quality control samples.

4) Preparing a characteristic peptide fragment mother solution: precisely measuring the characteristic peptide fragment stock solution ApoA1 pep 1200 mu L, ApoA1 pep 2200 mu L, ApoE pep 110 mu L, ApoE pep 210 mu L, ApoE pep 310 mu L, ApoJ pep 120 mu L and ApoJ pep 25 mu L in the step 2) respectively, putting the solution into a 10mL volumetric flask, adding the substituted matrix in the step 3) and diluting the solution to scale to prepare a characteristic peptide fragment mixed mother solution, wherein the corresponding concentrations are respectively as follows: 20. 20, 1, 2, 0.5. mu.g/mL. The mother liquor is frozen at the temperature below 70 ℃ before use.

5) Preparing a characteristic peptide fragment correction standard sample: precisely measuring 0, 10, 20, 50, 100, 200, 500 and 1000 mu L of the characteristic peptide fragment mother liquor in the step 4), respectively placing the mother liquor into a 10mL volumetric flask, adding the substituted matrix in the step 3), diluting to a scale, obtaining the prepared characteristic peptide fragment calibration standard samples STD 0-STD 7 with the concentrations shown in the table 2, subpackaging, labeling and finishing the operation.

6) Preparing a characteristic peptide fragment quality control sample: precisely measuring 30, 150 and 800 mu L of the characteristic peptide fragment mother liquor in the step 4), respectively placing the mother liquor into a 10mL volumetric flask, adding the substitute matrix in the step 3), diluting to scale, packaging the prepared characteristic peptide fragment quality control samples LQC, MQC and HQC with the concentrations shown in the table 2, labeling, and finishing the operation.

TABLE 2 preparation concentrations (ng/mL) of characteristic peptide fragment calibration standard and quality control sample

	ApoA1 pep1	ApoA1 pep2	ApoE pep1	ApoE pep2	ApoE pep3	ApoJ pep1	ApoJ pep2
								STD0	0	0	0	0	0	0	0
STD1	20	20	1	1	1	2	0.5
								STD2	40	40	2	2	2	4	1
STD3	100	100	5	5	5	10	2.5
								STD4	200	200	10	10	10	20	5
STD5	400	400	20	20	20	40	10
								STD6	1000	1000	50	50	50	100	25
STD7	2000	2000	100	100	100	200	50
								LQC	1600	1600	80	80	80	160	40
MQC	300	300	15	15	15	30	7.5
								HQC	60	60	3	3	3	6	1.5

2. Preparing a characteristic peptide fragment isotope internal standard solution: the endoplasmic internal standard information of the characteristic peptide segments of ApoA1, ApoE and ApoJ is shown in Table 1, and the characteristic peptide segment isotope internal standard product is synthesized. Precisely weighing a proper amount of each characteristic peptide isotope internal standard, dissolving the standard into 1mg/mL of each peptide isotope internal standard stock solution by using acetonitrile-water (v/v is 1:4) containing 0.1% of FA, and subpackaging and freezing the stock solution at the temperature of below 70 ℃ before use. Precisely measuring 100 mu L of each peptide isotope internal standard stock solution, placing the stock solution into a 100mL volumetric flask, dissolving and diluting the stock solution to a scale by using water containing 0.1 percent of FA, and preparing a mixed solution of 1 mu g/mL as the internal standard solution. Subpackaging, labeling and marking to finish the operation.

3. Preparation of Buffer 1(Buffer 1): 395.3mg of ammonium bicarbonate and 48g of urea are precisely weighed, put into a 100mL volumetric flask, and added with water to be dissolved and diluted to scale to prepare a solution containing 50mM ammonium bicarbonate and 8M urea. Subpackaging, labeling and marking to finish the operation.

4. Preparation of Buffer 2(Buffer 2): 395.3mg of ammonium bicarbonate is precisely weighed and placed in a 100mL volumetric flask, and water is added to dissolve and dilute the ammonium bicarbonate to a scale mark, so as to prepare a solution containing 50mM ammonium bicarbonate. Subpackaging, labeling and marking to finish the operation.

5. Preparation of treatment liquid A: accurately weighing 1542.5mg of DTT, placing the DTT in a 10mL volumetric flask, adding water to dissolve and dilute the DTT to a scale mark, and preparing a solution containing 1M DTT. Subpackaging, labeling and marking to finish the operation.

6. Preparation of treatment liquid B: 740mg of IAA is precisely weighed and placed in a 5mL volumetric flask, and water is added to dissolve and dilute the IAA to a scale mark, so as to prepare a solution containing 148mg/mL IAA. Subpackaging, labeling and marking to finish the operation. The preparation and the storage are carried out in dark.

Secondly, the using method of the kit is as follows:

1. calibration standards and quality control samples: accurately measuring 200 mu L of STD 0-STD 7 calibration standard samples, respectively adding 20 mu L of internal standard solution, mixing uniformly by vortex, and sampling 10 mu L of supernate to perform LC-MS/MS quantitative analysis.

2. Actual samples: precisely measuring 2 mu L of human plasma or serum, adding 2 mu L of the treatment solution A, adding 96 mu L of Buffer1, and incubating at 60 ℃ for 1 h; recovering to room temperature, adding 5 μ L of treatment solution B, and keeping at 25 deg.C in dark for 40 min; adding 700 mu L of Buffer2, whirling, adding 6 mu L of 1mg/mL Trypsin, and carrying out enzymolysis for 15h at 37 ℃; adding 10 mu L of FA to terminate enzymolysis, and adding 20 mu L of internal standard liquid to swirl; samples were applied to SPE cartridges (model: Oasis PRIME HLB Cartridge 1cc/30mg100 bx; or similar SPE cartridges that had been activated and equilibrated) and washed 3 times with 1mL of 0.1% TFA; the clean sample collection tube was replaced and eluted with 1mL acetonitrile-water (v/v ═ 4:1) solution containing 0.1% TFA; drying the eluent at 45 ℃ with nitrogen or freezing and drying; the blow-dried or freeze-dried sample can be placed in a refrigerator below-70 ℃ for a long time to be tested; 200 μ L of 0.1% FA was reconstituted and 10 μ L of the supernatant was injected for LC-MS/MS quantitative analysis.

Thirdly, the detection method comprises the following steps:

1. adapted for instruments

Liquid chromatography-tandem mass spectrometer (LC-MS/MS) equipped with electrospray ion source (ESI).

2. Sample requirement

Collecting venous whole blood sample, placing in anticoagulation and procoagulant tube, centrifugally separating blood plasma or serum, and testing as soon as possible after separation or placing in refrigerator below-20 deg.C for standby test.

3. Inspection method

Plasma or serum sample pretreatment:

precisely measuring 2 mu L of human plasma or serum, adding 2 mu L of the treatment solution A, adding 96 mu L of Buffer1, and incubating at 60 ℃ for 1 h; recovering to room temperature, adding 5 μ L of treatment solution B, and keeping at 25 deg.C in dark for 40 min; adding 700 mu L of Buffer2, whirling, adding 6 mu L of 1mg/mL Trypsin, and carrying out enzymolysis for 15h at 37 ℃; adding 10 mu L of FA to terminate enzymolysis, and adding 20 mu L of internal standard liquid to swirl; samples were applied to SPE cartridges (model: Oasis PRIME HLB Cartridge 1cc/30mg100 bx; or similar SPE cartridges that had been activated and equilibrated) and washed 3 times with 1mL of 0.1% TFA; the clean sample collection tube was replaced and eluted with 1mL acetonitrile-water (v/v ═ 4:1) solution containing 0.1% TFA; drying the eluent at 45 ℃ with nitrogen or freezing and drying; the blow-dried or freeze-dried sample can be placed in a refrigerator below-70 ℃ for a long time to be tested; 200 μ L of 0.1% FA was reconstituted and 10 μ L of the supernatant was injected for LC-MS/MS quantitative analysis.

Mass spectrum conditions:

an ion source: electrospray ion source (ESI), positive ion mode detection; ion ejection voltage: 5500V; temperature: 580 ℃; source gas 1(GS1, N)₂) Pressure: 60 psi.; gas 2(GS2, N)₂) Pressure: 60 psi.; gas curtain gas (N)₂) Pressure: 40 psi.; the scanning mode is Multiple Reaction Monitoring (MRM), and the parameters are shown in a table 3; collision gas (N)₂) Pressure: medium;

chromatographic conditions are as follows:

a chromatographic column: phenomenex Aeris WIDEPORE 3.6 μm XB-C18, 150X 2.1 mm;

mobile phase: 0.1% aqueous FA solution-0.1% FA acetonitrile, gradient elution, see table 4;

column temperature: 40 ℃; sample introduction amount: 10 μ L.

TABLE 3 MRM quantitative and qualitative correlation of ion Mass Spectrometry parameters

TABLE 4 gradient elution conditions

Time	Pump flow	0.1% aqueous FA solution%	0.1% FA acetonitrile%
				0	0.5	95	5
0.5	0.5	95	5
				2.5	0.5	75	25
3.5	0.5	55	45
				4.5	0.5	10	90
5.5	0.5	10	90
				5.6	0.5	95	5
8	0.5	95	5

And (5) sucking 10 mu L of each of the processed standard curve sample, the quality control sample and the sample solution to be detected to perform liquid chromatograph-mass spectrometer detection, and recording a chromatogram.

Analysis batch and quality control requirements:

one assay lot includes blank samples and zero concentration samples, including at least 6 concentration levels of calibration standards, at least 3 concentration levels of quality control samples (low, medium, high concentration double samples, or at least 5% of the total number of test samples, more in each case), and the test samples being analyzed. All samples (calibration standards, quality control and test samples) should be processed and extracted in the same sample batch in the order they are to be analyzed. Samples included in an assay batch are processed at the same time and sequentially by the same analyst, using the same reagents, maintaining consistent conditions. The quality control sample should be dispersed throughout the batch to ensure accuracy and precision throughout the analysis batch.

Calibration standards generally should be determined to recover concentrations within. + -. 15% of the indicated value, with a lower limit of quantitation within. + -. 20%. Not less than 6 calibration standards, at least 75% of which should meet these criteria. If one of the calibration standards does not meet the standard, the standard should be rejected, the standard curve without the standard calculated again, and regression analysis performed. The accuracy value of the quality control sample should be within. + -. 15% of the indicated value. At least 67% of the samples were quality controlled and at least 50% of the samples per concentration level should meet this criterion. In case these criteria are not met, the analysis batch should be rejected and the corresponding test sample should be re-extracted and analyzed.

4. And (4) calculating a result:

drawing a standard curve: and (3) drawing a standard curve by taking the labeled concentration of the 7 standard curve samples as an abscissa (x) and taking the ratio of the actually measured peak area of the standard curve sample to the peak area of each internal standard as an ordinate (y).

Fitting of standard curve equation: and performing linear regression on the marked concentration (x) by using the weighted least square method according to the ratio (y) of the actually measured peak area of the 7 standard curve samples to the peak area of each internal standard. Fitting a linear regression equation: y ═ ax + b, where a is the slope and b is the intercept, and the correlation coefficient (r) is calculated, r should not be less than 0.9900.

And (3) calculating the recovery rate: and substituting the ratio of the peak area of the target compound and the peak area of the internal standard, which are measured by the quality control sample, into the standard curve equation to calculate the concentration of the quality control sample. The calculation formula of the recovery rate of the quality control sample is as follows: the recovery (%) should be within 100 ± 15% of the measured/labeled concentration × 100.

And (3) calculating sample results: substituting the ratio of the peak area of the target compound of the sample to the peak area of the internal standard into a standard curve equation, calculating the concentration of each peptide segment of the sample, and converting the concentrations of apolipoprotein A, apolipoprotein E and apolipoprotein J according to the molar concentration.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The use, modifications and variations of the characteristic peptide fragments of the invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Beijing Chaoyang Hospital affiliated to capital medical university

<120> characteristic peptide fragment, detection method and kit

<130> CN20B13216A

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> ApoA1 pep1

<400> 1

Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys

1 5 10

<210> 2

<211> 9

<212> PRT

<213> ApoA1 pep2

<400> 2

Leu Ser Pro Leu Gly Glu Glu Met Arg

1 5

<210> 3

<211> 9

<212> PRT

<213> ApoE pep1

<400> 3

Leu Gly Pro Leu Val Glu Gln Gly Arg

1 5

<210> 4

<211> 15

<212> PRT

<213> ApoE pep2

<400> 4

Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg

1 5 10 15

<210> 5

<211> 9

<212> PRT

<213> ApoE pep3

<400> 5

Leu Gln Ala Glu Ala Phe Gln Ala Arg

1 5

<210> 6

<211> 23

<212> PRT

<213> ApoJ pep1

<400> 6

Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly

1 5 10 15

Val Thr Glu Val Val Val Lys

20

<210> 7

<211> 10

<212> PRT

<213> ApoJ pep2

<400> 7

Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys

1 5 10

Claims (10)

1. The characteristic peptide fragment is characterized by at least comprising peptide fragments with amino acid sequences of QGLLPVLESFK, LSPLGEEMR, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, VTTVASHTSDSDVPSGVTEVVVK and TLLSNLEEAK respectively.

2. A detection method comprising using an isotopically labeled peptide fragment corresponding to the characteristic peptide fragment of claim 1 as an internal standard when detecting apolipoprotein A, apolipoprotein E and apolipoprotein J simultaneously.

3. The detection method of claim 2, comprising:

preparing an isotope labeled peptide fragment internal standard solution;

preparing a correction standard sample of the characteristic peptide fragment by using a substitute matrix;

treating a sample to be detected with a dithiothreitol solution, an iodoacetamide solution and trypsin in sequence, adding an isotope labeled peptide internal standard solution, loading onto a column, eluting, drying by nitrogen blowing or freezing, drying, and redissolving to obtain a sample solution to be detected;

adding an isotope labeled peptide fragment internal standard solution into the calibration standard sample, analyzing by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide fragment and the peak area of the corresponding isotope labeled peptide fragment, and establishing a standard curve taking the peak area ratio and the concentration as coordinates;

and analyzing the sample solution to be detected by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide segment and the peak area of the corresponding isotope labeled peptide segment, substituting the ratio of the peak areas into a standard curve, and calculating the concentrations of the apolipoprotein A, the apolipoprotein E and the apolipoprotein J.

4. The detection method of claim 3, further comprising:

preparing a quality control sample of the characteristic peptide segment by using a substitute matrix, adding an isotope labeled peptide segment internal standard solution into the quality control sample, analyzing by using a liquid chromatography-mass spectrometry instrument, recording the peak area of the characteristic peptide segment and the peak area of the corresponding isotope labeled peptide segment, substituting the ratio of the peak areas into a standard curve, and calculating the recovery rate value of the quality control sample.

5. The detection method of claim 3, wherein the surrogate matrix is prepared by a method comprising:

taking bovine albumin, sequentially treating with dithiothreitol solution, iodoacetamide solution and trypsin, loading onto column, eluting, drying with nitrogen gas or freeze drying, and redissolving to obtain the substitute matrix.

6. The detection method of claim 3, wherein the liquid chromatography-mass spectrometry instrument is an LC-MS/MS;

the mass spectrometry conditions include: the ion source is an electrospray ion source, the positive ion mode detection is adopted, and the scanning mode is multiple reaction monitoring.

7. The assay of claim 6, wherein the parameters for multiplex reaction monitoring comprise:

wherein VTTVASHTSDSDVPSGVTEVVVK, TLLSNLEEAK, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, QGLLPVLESFK and LSPLGEEMR respectively represent corresponding isotopically labeled peptide fragments of characteristic peptide fragment VTTVASHTSDSDVPSGVTEVVVK, TLLSNLEEAK, LGPLVEQGR, AATVGSLAGQPLQER, LQAEAFQAR, QGLLPVLESFK, LSPLGEEMR.

8. A kit, characterized in that it comprises:

an internal standard solution prepared from isotope-labeled peptide fragments corresponding to the characteristic peptide fragments in claim 1;

a calibration standard sample prepared from a substitute matrix and the characteristic peptide fragment;

dithiothreitol solution and iodoacetamide solution for treating the sample to be tested.

9. The kit of claim 8, further comprising:

and the quality control sample is prepared from a substitute matrix and the characteristic peptide fragment, and at least comprises three different concentrations, namely high concentration, medium concentration and low concentration.

10. The kit of claim 8, further comprising:

the buffer solution 1 is prepared from ammonium bicarbonate, urea and water, and the buffer solution 2 is prepared from ammonium bicarbonate and water.

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