CN108398503A - A kind of liquid chromatography mass detection method of lactoferrin - Google Patents
A kind of liquid chromatography mass detection method of lactoferrin Download PDFInfo
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- CN108398503A CN108398503A CN201810254892.6A CN201810254892A CN108398503A CN 108398503 A CN108398503 A CN 108398503A CN 201810254892 A CN201810254892 A CN 201810254892A CN 108398503 A CN108398503 A CN 108398503A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention relates to a kind of liquid chromatography mass assay methods of lactoferrin, including：Sample to be tested and lactoferrin standard items are subjected to trypsin digestion processing respectively, the sample to be tested test liquid containing special peptide fragment, the processing test solution of the lactoferrin standard working solution containing special peptide fragment is made；Special peptide fragment standard working solution is prepared with special peptide fragment standard items；Carry out high performance liquid chromatography Mass Spectrometer Method.The present invention digests controlled enzymatic hydrolysis coefficient simultaneously with special peptide fragment quantified by external standard method, lactoferrin standard, being capable of non denatured in determination sample and thermal denaturation bovine lactoferrin content.
Description
Technical field
The present invention relates to the assay methods of lactoferrin, belong to dairy products detection technique field.
Background technology
Lactoferrin is a kind of natural, glycoprotein with immune function, and from transferrins transformation, crystal is in red
Color is about made of 700 amino acid residues, molecular weight 80ku, isoelectric point 8.7, can be combined with iron invertibity, mainly
It is present in milk, while also has presence in tear, saliva, blood plasma, bile, pancreatic juice and neutrophil cell.Lactoferrin
Content is higher in people's colostrum, a concentration of 6~8mg/mL；A concentration of 1~2mg/mL in Bovine Colostrum, and in normal breast content compared with
It is low, a concentration of 1~2mg/mL during people is often newborn, a concentration of 0.02~0.35mg/mL during milk cow is often newborn.In different nursery phase milk
Lactoferrin content constantly changes.
Lactoferrin belongs to the active material of innate immune system, also by as a kind of functional component-reactive protein,
China, lactoferrin starting is comparatively later, and its effect is mainly reflected in antibacterial aspect, but in addition to this, lactoferrin
Also with bactericidal effect, iron binding capacity and to the facilitation of iron absorption and radiation resistance etc..Lactoferrin is as work(
Energy property ingredient, has been applied to many fields, such as food, medicine, dentistry etc..
So far, the standard determination method of lactoferrin is promulgated not yet both at home and abroad.Lactoferrin qualitative and quantitative
Detection method includes mainly enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis method
(SDS-PAGE) and reversed-phased high performace liquid chromatographic (RP-HPLC) etc..ELISA method is to detect specific protein to have an efficacious prescriptions
Method has many advantages, such as that quick, sensitive, easy, carrier is easy to standardization.In conjunction with SDS-PAGE, can be used for for specific antigen
Qualitative and quantitative detection.The lactoferrin content in fresh milk and milk products is often detected in practice using such method.
Enzyme-linked immunosorbent assay (enzyme-linkedimmunosorbent assay, ELISA) can be divided into measurement
The indirect method of antibody, 3 kinds of double-antibody method and competition law for measuring antigen.The detection of lactoferrin content generally uses in sample
Double antibody sandwich method.ELISA method measure lactoferrin content have quick and precisely, detection is for normal breast, colostrum, milk powder, milk
The detection of the lactoferrin in dairy products such as junket.But this method also has certain defect, needs to carry out sample before determination sample more
Secondary dilution, and the method used kit is costly.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (sodium dodecyl suate-
Polyacrylamide gelelectrophoresis, SDS-PAGE) it is that one kind being commonly used in purifying and qualitative analysis albumen
The bioanalytical method of matter, the application that lactoferrin is quantitatively detected in relation to it have been reported.Newborn iron egg is detached using electrophoresis
Have many advantages, such as that equipment is simple, low-cost, operating method is simple in vain, but it is there is also some disadvantages, such as time-consuming, should not examine
Survey that batch samples, quantitatively detection lactoferrin process is complex etc..
Reversed-phased high performace liquid chromatographic (reversed phase highperformance liquid
Chromatography, RP-HPLC) in the research in relation to lactoferrin measurement at home and abroad, both about utilizing positive
The report of high effective liquid chromatography for measuring lactoferrin also has the relevant report that lactoferrin is measured using RP-HPLC methods, at present
Relatively conventional using RP-HPLC methods, the optimization about liquid chromatography for measuring condition has existed many researchs.
Random immunodiffusion (random immune diffusion, RID) i.e. antigen, antibody is in Ago-Gel
Free diffusing, the two are met at the appropriate place of ratio, and precipitation reaction occurs.The sediment of reaction is because its particle is larger, in gel
It no longer spreading, forms sealed Belt, the diameter of sealed Belt and the concentration of lactoferrin are proportional, are dyed sealed Belt with dyeing liquor,
The diameter for accurately measuring ring calculates the concentration of lactoferrin in test specimen by canonical plotting.
Spectrophotometry is the trap by measuring substance light in certain wave strong point or a wavelength range, to the object
The method that matter carries out qualitative and quantitative analysis.Requirement with spectrophotometry measurement lactoferrin concentration to instrument and equipment is relatively low,
The content that lactoferrin can rapidly be measured, during being isolated and purified factory it is quick detection or to detection tie
The analysis of the not high sample of fruit accuracy requirement, but its accuracy is poor.
In view of this, special propose the present invention.
Invention content
The object of the present invention is to provide a kind of liquid chromatography mass assay methods of lactoferrin, can rapidly and accurately survey
Fixed non denatured and thermal denaturation bovine lactoferrin content, compensates for other methods in detection range, applicability or detection efficiency
Defect.
The present invention adopts the following technical scheme that：
A kind of liquid chromatography mass assay method of lactoferrin, including：By sample to be tested and lactoferrin standard items point
Not carry out trypsin digestion processing, the sample to be tested test liquid containing special peptide fragment, the lactoferrin mark containing special peptide fragment is made
Quasi- working solution handles test solution；Special peptide fragment standard working solution is prepared with special peptide fragment standard items；Carry out high performance liquid chromatography mass spectrum
Detection.
If it is no it is special indicate, the amino acid sequence of special peptide fragment of the present invention is VDSALYLGSR.
Further, the sample to be tested test liquid containing special peptide fragment, the work of the lactoferrin standard containing special peptide fragment
Liquid handles test solution and is made by trypsin digestion；Specifically, sample to be tested or lactoferrin standard items can be used carbonic acid successively
Hydrogen ammonium, dithiothreitol (DTT), iodo-acetamide processing, then again existing for calcium chloride under the conditions of handled with trypsin digestion.
Specifically, the preparation method of the sample to be tested test liquid containing special peptide fragment includes：Sample is made in sample to be tested
Ammonium hydrogen carbonate or its solution is added in product solution, and dithiothreitol (DTT) or its solution is added in mixing, in 40-60 DEG C (preferably 50 DEG C) perseverance
Temperature reaction 30-60min, cooling；Iodo-acetamide or its solution is added, the static 30-40min in dark place, is added calcium chloride or its is molten
Liquid and trypsase or its solution, enzymolysis (are preferable over 37 DEG C of constant temperature to digest 4-6 hours) completely, and formic acid, the static 30- of room temperature is added
60min finally adds water constant volume, 0.22 μm of filter membrane is crossed after mixing, you can.
When sample to be tested is solid-state (such as solid-state dairy products milk powder), the sample of a concentration of 0.5-10g/100mL can be made into
Product solution；When sample to be tested is liquid (such as liquid diary product milk), it is 0.5-10g/100mL that can be made into solid concentration
Sample solution.Or further appropriate dilution.
Preferably, sample to be tested is configured to the sample solution that total protein concentration or solid concentration are about 1.0mg/mL,
It may generally be the sample solution of total protein concentration or solid concentration 0.5-1.5mg/mL.
Specifically, the preparation method of the sample to be tested test liquid containing special peptide fragment includes：Sample to be tested is made always
Albumen concentration or the sample solution that solid concentration is 2-10g/100mL, draw 100-300 μ L sample solution, 150-200 are added
The ammonium bicarbonate soln of a concentration of 500mmol/L of μ L, mixing, the dithiothreitol (DTT) that a concentration of 500mmol/L of 10-20 μ L are added are molten
Liquid, 50 DEG C of isothermal reaction 30-60min take out cooling, and the iodoacetamido amine aqueous solution of a concentration of 500mmol/L of 40-50 μ L is added,
The calcium chloride solution and a concentration of 100 μ g/mL pancreases of 30-60 μ L of 10 a concentration of 100mmol/L of μ L is added in the static 30-40min in dark place
Protein enzyme solution, 37 DEG C of constant temperature digest 4-6 hours, and 10-20 μ L formic acid is added after taking-up, and the static 30-60min of room temperature finally adds
Water, it is 1.0mL to make enzymolysis liquid total volume, and 0.22 μm of filter membrane is crossed after mixing, you can.
Specifically, the preparation method of the lactoferrin standard working solution processing test solution containing special peptide fragment includes：It takes suitable
Lactoferrin standard items are measured, certain density standard working solution is configured to；By with sample to be tested of the above-mentioned preparation containing special peptide fragment
The same procedure of test liquid is handled.
Specifically, the preparation method of the lactoferrin standard working solution processing test solution containing special peptide fragment includes：It takes suitable
Lactoferrin standard items are measured, standard working solution (such as a concentration of 10-500 μ g/mL, the preferred concentration of a series of concentration are configured to
For 10-500 μ g/mL)；The 100-300 μ L standard working solutions are drawn, the bicarbonate of a concentration of 500mmol/L of 150-200 μ L is added
Ammonium salt solution, mixing, the dithiothreitol (DTT) solution of a concentration of 500mmol/L of addition 10-20 μ L, 50 DEG C of isothermal reaction 30-60min,
Cooling is taken out, the iodoacetamido amine aqueous solution of a concentration of 500mmol/L of 40-50 μ L is added, 10 μ L are added in the static 30-40min in dark place
A concentration of 100 μ g/mL trypsin solutions of calcium chloride solution and 30-60 μ L of a concentration of 100mmol/L, 37 DEG C of constant temperature digest 4-
6 hours, 10-20 μ L formic acid is added after taking-up, the static 30-60min of room temperature finally adds water, and it is 1.0mL to make enzymolysis liquid total volume,
0.22 μm of filter membrane is crossed after mixing, you can.
Specifically, the preparation method of the special peptide fragment standard working solution includes：Special peptide fragment standard items are accurately weighed, are matched
It is set to a series of special peptide fragment standard working solution of concentration, such as concentration range can be 100-550ng/mL.
Specifically, liquid phase chromatogram condition：
Using silylation C18 chromatographic columnsOr quite.
Mobile phase：0.1% trifluoroacetic acid aqueous solution of A phases or 0.1% aqueous formic acid；B phases acetonitrile or 0.1% formic acid acetonitrile
Solution；
Flow velocity：0.25-0.35mL/min；
Column temperature：35-45℃；
Gradient elution see the table below：
| Time/min | A phases | B phases |
| 0-1.2 | 95% | 5% |
| 1.2-2.0 | 95-80% | 20-5% |
| 2.0-3.8 | 80-70% | 30-20% |
| 3.8-4.0 | 70-0% | 100-30% |
| 4.0-4.8 | 0% | 100% |
| 4.8-5.0 | 0-95% | 5-100% |
| 5.0-7.0 | 95% | 5% |
Preferably, condition of gradient elution is as follows：
| Time/min | A phases | B phases/% |
| 0-1.2 | 95% | 5% |
| 2.0 | 80% | 20% |
| 3.8 | 70% | 30% |
| 4.0-4.8 | 0% | 100% |
| 4.8-5.0 | 95% | 5% |
| 5.0-7.0 | 95% | 5% |
Preferably, mobile phase：0.1% aqueous formic acid of A phases；B phase acetonitriles.
Mass Spectrometry Conditions：Capillary voltage 3.0-3.5kv, orifice potential 30-35kv, 450-550 DEG C of desolventizing temperature, precipitation
Agent throughput 750-850L/min, cone hole backflow airflow amount 30L/hr collide chamber pressure 3.0 × 10-3Mbar, low side resolution ratio 1:
2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.5, low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V ion
Energy 2:1.0, ion source temperature：150 DEG C, extractor pressure：3.0V, entrance lens voltage：0.5V collides gradient：1.0.
The MS detection parameters：
Wherein, parent ion [M+2H]2+540.9；Daughter ion 595.3 is quota ion；Daughter ion 319.3 is qualitative ion.
Preferably, Mass Spectrometry Conditions：Capillary voltage 3.5kv, orifice potential 35kv, 500 DEG C of desolventizing temperature, desolventizing gas
Flow 800L/min, cone hole backflow airflow amount 30L/hr collide chamber pressure 3.0 × 10-3Mbar, low side resolution ratio 1:2.5V is high
Hold resolution ratio 1:15.0V, ion energy 1:0.5, low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V, ion energy 2:
1.0, ion source temperature：150 DEG C, extractor pressure：3.0V, entrance lens voltage：0.5V collides gradient：1.0.
Qualitative detection：As parent ion [M+2H]2+540.9 is (qualitative with daughter ion 595.3 (quota ion), daughter ion 319.3
Ion) exist simultaneously and it is consistent with the special appearance time of peptide fragment standard working solution when, then judge in sample to be tested judge contain
Lactoferrin；When not being detected simultaneously by parent ion [M+2H]2+540.9 with daughter ion 595.3 (quota ion), daughter ion 319.3
When (qualitative ion), then judge to judge to be free of lactoferrin in sample to be tested.
Quantitative detection：The enzymolysis coefficient correction sample Degree of Enzymatic Hydrolysis of test solution, profit are handled according to lactoferrin standard working solution
It is detected with quantified by external standard method.
Specifically, quantitative detect includes：
According to liquid chromatogram, the mass spectrogram of special peptide fragment standard working solution, with the peak area of special peptide fragment quota ion
For abscissa, a concentration of ordinate of special peptide fragment standard working solution, special peptide fragment standard curve is drawn；
Liquid chromatogram, the mass spectrogram that test solution is handled according to lactoferrin standard working solution, with special peptide fragment quota ion
To chromatographic peak area and above-mentioned special peptide fragment standard curve obtain lactoferrin standard working solution processing test solution in special peptide fragment
Concentration；
It is dense according to special peptide fragment in the concentration of lactoferrin standard working solution, lactoferrin standard working solution processing test solution
Degree calculates lactoferrin according to following formula and digests coefficient：
CLactoferrin=K × CSpecial peptide fragment, wherein K is enzymolysis coefficient；
The Degree of Enzymatic Hydrolysis that lactoferrin in coefficient correction sample to be tested test liquid is digested with lactoferrin, using above-mentioned special
Peptide fragment standard curve calculates the content of lactoferrin in sample to be tested (preferably with external standard method).
Detection method suitable application area is wide, can be used for the detection of varieties of food items, drug, biological products etc., especially suitable
Detection for dairy products.
The beneficial effects of the present invention are：
The liquid chromatography mass assay method for establishing lactoferrin, thermal denaturation breast iron cannot be measured by solving current method
The defect of albumen.
The method of the present invention has high-specificity, the low feature of cost of determination.Dairy products detection field lactoferrin is quantified
Measurement is of great significance.
Description of the drawings
The special peptide fragment standard working solution mass spectrograms of 1 a concentration of 304.74ng/mL of Fig. 1 embodiments；
1 a concentration of 49.8 μ g/mL lactoferrin standard working solutions of Fig. 2 embodiments handle test solution mass spectrogram；
1 sample to be tested test liquid mass spectrogram of Fig. 3 embodiments；
1 sample to be tested test liquid mass spectrogram of Fig. 4 embodiments；
1 lactoferrin standard working solution of Fig. 5 embodiments handles lactoferrin and special peptide fragment relation curve in test solution；
1 sample lactoferrin liquid chromatogram of Fig. 6 comparative examples.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is not specified in embodiment specific
Technology or condition person carry out according to technology or condition described in document in the art, or according to product description.It is used
Production firm person is not specified in reagent or instrument, is the conventional products that can be commercially available by regular distributor.
Key instrument：Liquid chromatogram level four bars mass spectrometer, charged spray ion source, WATERS, UPLC I-CLASS
XEVO TQ。
Main agents：
Ammonium bicarbonate soln：500mmol/L；
Dithiothreitol (DTT) solution：500mmol/L；
Iodoacetamido amine aqueous solution：500mmol/L；
Calcium chloride solution：100mmol/L；
Bovine trypsin solution：100μg/mL.
Lactoferrin standard items, purity >=95%, purchased from Japan and light (WAKO) company；
The special peptide fragment standard items of bovine lactoferrin, amino acid sequence VDSALYLGSR, purity >=95%, by Ningbo health shellfish
Biochemical Co., Ltd provides.
The detection of lactoferrin in 1 milk powder of embodiment
1) pretreatment of sample to be tested
Powdered milk sample 10g is weighed, is dissolved with 35 DEG C of warm water water bath sonicators, 100mL is settled to after cooling, as powdered milk sample
Solution.
The above-mentioned powdered milk sample solution of 200 μ L is drawn in small test tube, 180 μ L ammonium bicarbonate solns are added, mixing is added 15
μ L dithiothreitol (DTT) solution, 50 DEG C of isothermal reaction 30min take out cooling, and 45 μ L iodoacetamido amine aqueous solutions are added, and dark place is static
10 μ L calcium chloride solutions and 40 μ L trypsin solutions are added in 30min, and 37 DEG C of constant temperature digest 4 hours, and 10 μ L first are added after taking-up
Acid, the static 30min of room temperature, is eventually adding suitable quantity of water, and it is 1.0mL to make enzymolysis liquid total volume, crosses 0.22 μm of filter membrane after mixing, as
Sample to be tested test liquid containing special peptide fragment is measured for liquid chromatography mass.
2) it prepares the lactoferrin standard working solution containing special peptide fragment and handles test solution
Lactoferrin standard items are taken, using water as solvent, compound concentration is 13.67 μ g/mL, 25.54 μ g/mL, 49.81 respectively
The lactoferrin standard working solution of μ g/mL, 93.63 μ g/mL, 118.48 μ g/mL.The 200 μ L lactoferrin standard works are taken respectively
Make liquid, enzymolysis processing is carried out by the identical method of step 1), obtains lactoferrin standard working solution processing test solution.
3) special peptide fragment standard working solution is prepared
The special peptide fragment standard items (amino acid sequence VDSALYLGSR) of bovine lactoferrin accurately are weighed, using water as solvent,
First be configured to the storing solution of a concentration of 500ng/mL, be then diluted to respectively again a concentration of 101.58ng/mL, 203.16ng/mL,
The special peptide fragment standard working solution of 304.74ng/mL, 406.32ng/mL, 507.90ng/mL.
4) with Liquid Chromatography-Tandem Mass Spectrometry respectively to above-mentioned sample to be tested test liquid, lactoferrin standard working solution at
Manage test solution, special peptide fragment standard working solution carries out liquid chromatography mass detection.
Liquid phase chromatogram condition：
Using silylation C18 chromatographic columns
Mobile phase：0.1% aqueous formic acid of A phases, B phase acetonitriles；
Flow velocity：0.3mL/min；
Column temperature：40℃；
Gradient elution：
| Time/min | A phases | B phases/% |
| 0-1.2 | 95% | 5% |
| 2.0 | 80% | 20% |
| 3.8 | 70% | 30% |
| 4.0 | 0% | 100% |
| 4.0-4.8 | 0% | 100% |
| 5.0 | 95% | 5% |
| 5.0-7.0 | 95% | 5% |
Mass Spectrometry Conditions：Capillary voltage 3.5kv, orifice potential 35kv, 500 DEG C of desolventizing temperature, desolventizing gas flow
800L/min, cone hole backflow airflow amount 30L/hr collide chamber pressure 3.0 × 10-3Mbar, low side resolution ratio 1:2.5V, high-end point
Resolution 1:15.0V, ion energy 1:0.5, low side resolution ratio 2:2.8V high-end resolution ratio 2:15.0V, ion energy 2:1.0, from
Source temperature：150 DEG C, extractor pressure：3.0V, entrance lens voltage：0.5V collides gradient：1.0.
The MS detection parameters
Testing result (liquid chromatogram, mass spectrogram) is shown in Fig. 1-Fig. 4.
5) qualitative detection：According to liquid chromatogram, mass spectrogram it is found that parent ion [M+2H]2+540.9 with daughter ion 595.3
(quota ion), daughter ion 319.3 (qualitative ion) exist simultaneously and consistent with the special appearance time of peptide fragment standard working solution,
To judge to judge to contain lactoferrin in sample to be tested.
6) quantitative detection：
Make special peptide fragment standard curve：According to above-mentioned concentration be respectively 101.58ng/mL, 203.16ng/mL,
Liquid chromatogram, the mass spectrogram of the special peptide fragment standard working solution of 304.74ng/mL, 406.32ng/mL, 507.90ng/mL, with fixed
The peak area for measuring ion is a concentration of ordinate of abscissa, special peptide fragment standard working solution, draws special peptide fragment standard curve,
It is Y=0.0167X -1.8344, coefficient R to obtain equation of linear regression2=0.9973.Wherein, Y indicates special peptide fragment concentration
(ng/mL), X indicates quota ion peak area.
Lactoferrin digests coefficient and determines：According to above-mentioned a concentration of 13.67 μ g/mL, 25.54 μ g/mL, 49.81 μ g/mL,
Liquid chromatogram, mass spectrogram (the special peptide of the lactoferrin standard working solution processing test solution of 93.63 μ g/mL, 118.48 μ g/mL
The chromatographic peak area of section quota ion pair), the processing examination of lactoferrin standard working solution is obtained by above-mentioned special peptide fragment standard curve
Special peptide fragment concentration in liquid,
With a concentration of abscissa of lactoferrin standard working solution, lactoferrin standard working solution handles special peptide in test solution
A concentration of ordinate of section calculates lactoferrin according to following formula and digests coefficient：
CLactoferrin=K × CSpecial peptide fragment, wherein K is enzymolysis coefficient.
It is 1.2767 to acquire enzymolysis COEFFICIENT K, related coefficient R2=0.99.
That is CLactoferrin=1.2767 × CSpecial peptide fragment
Lactoferrin is shown in Fig. 5 with special peptide fragment relation curve in lactoferrin standard working solution processing test solution.
The Degree of Enzymatic Hydrolysis that lactoferrin in coefficient correction sample to be tested test liquid is digested with lactoferrin, using above-mentioned special
Peptide fragment standard curve calculates the content of lactoferrin in sample to be tested with external standard method, to calculate lactoferrin in powdered milk sample
Content is 72.63mg/100g.
Comparative example 1
Take powdered milk sample 10g same as Example 1,100mL be settled to after being dissolved in water, respectively at 40 DEG C, 60 DEG C, 80
Then DEG C heating water bath 1 hour measures lactoferrin content according to 1 identical method of embodiment, as a result as follows：
Upper table the result shows that, the milk powder measurement result of the processing of above-mentioned heating water bath and 1 no heat treatment of embodiment is without significance difference
It is different.
Comparative example 2
Powdered milk sample 2g same as Example 1 is taken, 10mL is settled to after being dissolved in water, respectively at 40 DEG C, 60 DEG C, 80 DEG C
Heating water bath 1 hour measures lactoferrin content using 1 method of patent CN 102590418A embodiments.The result shows that 40 DEG C
1 hour lactoferrin content of heating water bath falls to 85% without significant changes, 60 DEG C of heating water baths, 1 hour lactoferrin content,
80 DEG C of heating water baths, 1 hour lactoferrin content falls to 50%.Illustrate that this method is better than in the measurement of heat treatment lactoferrin
Liquid phase process.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing City Nutrient Source Inst
<120>A kind of liquid chromatography mass detection method of lactoferrin
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Val Asp Ser Ala Leu Tyr Leu Gly Ser Arg
1 5 10
Claims (10)
1. a kind of liquid chromatography mass assay method of lactoferrin, which is characterized in that including：By sample to be tested and lactoferrin
Standard items carry out trypsin digestion processing respectively, and the sample to be tested test liquid containing special peptide fragment, the breast containing special peptide fragment is made
Ferritin standard working solution handles test solution；Special peptide fragment standard working solution is prepared with special peptide fragment standard items；Carry out efficient liquid phase
Chromatographic mass spectrometry detects.
2. according to the method described in claim 1, it is characterized in that, the amino acid sequence of the special peptide fragment is
VDSALYLGSR。
3. method according to claim 1 or 2, which is characterized in that the sample to be tested test liquid containing special peptide fragment contains
Specifically the preparation method of the lactoferrin standard working solution processing test solution of peptide fragment includes：By sample to be tested, lactoferrin standard items
Respectively successively use ammonium hydrogen carbonate, dithiothreitol (DTT), iodo-acetamide processing, then again existing for calcium chloride under the conditions of use pancreas egg
White enzyme enzymolysis processing.
4. method according to claim 1 or 2, which is characterized in that the sample to be tested test liquid containing special peptide fragment
Preparation method includes：Sample solution is made in sample to be tested, ammonium hydrogen carbonate or its solution is added, dithiothreitol (DTT) is added in mixing
Or its solution, it is cooling in 40-60 DEG C of isothermal reaction 30-60min；Iodo-acetamide or its solution, the static 30- in dark place is added
Calcium chloride or its solution and trypsase or its solution is added in 40min, and enzymolysis is complete, and formic acid, the static 30- of room temperature is added
60min finally adds water constant volume, 0.22 μm of filter membrane is crossed after mixing, you can；
Preferably, the preparation method of the sample to be tested test liquid containing special peptide fragment includes：Total protein is made in sample to be tested
Concentration or the sample solution that solid concentration is 2-10g/100mL, draw 100-300 μ L sample solution, it is dense that 150-200 μ L are added
Degree is the ammonium bicarbonate soln of 500mmol/L, and the dithiothreitol (DTT) solution of a concentration of 500mmol/L of 10-20 μ L is added in mixing,
50 DEG C of isothermal reaction 30-60min take out cooling, and the iodoacetamido amine aqueous solution of a concentration of 500mmol/L of 40-50 μ L, dark place is added
The calcium chloride solution and a concentration of 100 μ g/mL tryptoses of 30-60 μ L of 10 a concentration of 100mmol/L of μ L is added in static 30-40min
Enzyme solutions, 37 DEG C of constant temperature digest 4-6 hours, and 10-20 μ L formic acid is added after taking-up, and the static 30-60min of room temperature finally adds water, makes
Enzymolysis liquid total volume is 1.0mL, and 0.22 μm of filter membrane is crossed after mixing, you can.
5. according to claim 1-4 any one of them methods, which is characterized in that the lactoferrin standard containing special peptide fragment
Working solution processing test solution preparation method include：Appropriate lactoferrin standard items are taken, a series of standard work of concentration is configured to
Liquid；The absorption 100-300 μ L standard working solutions, the ammonium bicarbonate soln of a concentration of 500mmol/L of addition 150-200 μ L, mixing,
The dithiothreitol (DTT) solution of a concentration of 500mmol/L of 10-20 μ L is added, 50 DEG C of isothermal reaction 30-60min take out cooling, are added
It is a concentration of that 10 μ L are added in the iodoacetamido amine aqueous solution of a concentration of 500mmol/L of 40-50 μ L, the static 30-40min in dark place
A concentration of 100 μ g/mL trypsin solutions of calcium chloride solution and 30-60 μ L of 100mmol/L, 37 DEG C of constant temperature digest 4-6 hours,
10-20 μ L formic acid is added after taking-up, the static 30-60min of room temperature finally adds water, makes enzymolysis liquid total volume for 1.0mL, after mixing
Cross 0.22 μm of filter membrane, you can.
6. according to claim 1-5 any one of them methods, which is characterized in that liquid phase chromatogram condition includes：
Mobile phase：0.1% trifluoroacetic acid aqueous solution of A phases or 0.1% aqueous formic acid；B phases acetonitrile or 0.1% formic acid acetonitrile are molten
Liquid；
Gradient elution is as follows：
And/or the MS detection parameters：
7. according to the method described in claim 6, it is characterized in that, gradient elution is as follows：
Preferably, mobile phase：0.1% aqueous formic acid of A phases；B phase acetonitriles.
8. according to claim 1-7 any one of them methods, which is characterized in that further include qualitative detection：As parent ion [M+
2H]2+540.9 with daughter ion 595.3, daughter ion 319.3 exist simultaneously and with the appearance time one of special peptide fragment standard working solution
When cause, then judge to judge to contain lactoferrin in sample to be tested；When not being detected simultaneously by parent ion [M+2H]2+540.9 with son from
When son 595.3, daughter ion 319.3, then judge to judge to be free of lactoferrin in sample to be tested.
9. according to claim 1-8 any one of them methods, which is characterized in that further include quantitative detection：
It is cross with the peak area of special peptide fragment quota ion according to liquid chromatogram, the mass spectrogram of special peptide fragment standard working solution
A concentration of ordinate of coordinate, special peptide fragment standard working solution draws special peptide fragment standard curve；
Liquid chromatogram, the mass spectrogram that test solution is handled according to lactoferrin standard working solution, with special peptide fragment quota ion pair
Chromatographic peak area and the special peptide fragment standard curve obtain special peptide fragment concentration in lactoferrin standard working solution processing test solution；
According to special peptide fragment concentration, root in the concentration of lactoferrin standard working solution, lactoferrin standard working solution processing test solution
Lactoferrin, which is calculated, according to following formula digests coefficient：
CLactoferrin=K × CSpecial peptide fragment, wherein K is enzymolysis coefficient；
The Degree of Enzymatic Hydrolysis that lactoferrin in coefficient correction sample to be tested test liquid is digested with lactoferrin, using the special peptide fragment
Standard curve calculates the content of lactoferrin in sample to be tested.
10. according to claim 1-9 any one of them methods, which is characterized in that including：
1) pretreatment of sample to be tested
Powdered milk sample 10g is weighed, is dissolved with 35 DEG C of warm water water bath sonicators, 100mL is settled to after cooling, it is molten as powdered milk sample
Liquid；
The above-mentioned powdered milk sample solution of 200 μ L is drawn in small test tube, 180 μ L ammonium bicarbonate solns are added, 15 μ L bis- are added in mixing
Sulphur threose alcoholic solution, 50 DEG C of isothermal reaction 30min take out and cool down, 45 μ L iodoacetamido amine aqueous solutions of addition, the static 30min in dark place,
10 μ L calcium chloride solutions and 40 μ L trypsin solutions are added, 37 DEG C of constant temperature digest 4 hours, and 10 μ L formic acid, room are added after taking-up
The static 30min of temperature, is eventually adding suitable quantity of water, and it is 1.0mL to make enzymolysis liquid total volume, crosses 0.22 μm of filter membrane after mixing, as contains spy
The sample to be tested test liquid of different peptide fragment is measured for liquid chromatography mass；
Ammonium bicarbonate soln：500mmol/L；Dithiothreitol (DTT) solution：500mmol/L；Iodoacetamido amine aqueous solution：500mmol/L；
Calcium chloride solution：100mmol/L；Bovine trypsin solution：100μg/mL；
2) it prepares the lactoferrin standard working solution containing special peptide fragment and handles test solution
Lactoferrin standard items are taken, using water as solvent, compound concentration is 13.67 μ g/mL, 25.54 μ g/mL, 49.81 μ g/ respectively
The lactoferrin standard working solution of mL, 93.63 μ g/mL, 118.48 μ g/mL；200 μ L lactoferrin standard work is taken respectively
Liquid carries out enzymolysis processing by the identical method of step 1), obtains lactoferrin standard working solution processing test solution；
3) special peptide fragment standard working solution is prepared
Accurately weigh the special peptide fragment standard items of bovine lactoferrin is first configured to the deposit of a concentration of 500ng/mL using water as solvent
Liquid, be then diluted to respectively again a concentration of 101.58ng/mL, 203.16ng/mL, 304.74ng/mL, 406.32ng/mL,
507.90ng/mL special peptide fragment standard working solution；
4) with Liquid Chromatography-Tandem Mass Spectrometry respectively to above-mentioned sample to be tested test liquid, the processing examination of lactoferrin standard working solution
Liquid, special peptide fragment standard working solution carry out liquid chromatography mass detection；
Liquid phase chromatogram condition：
Using silylation C18 chromatographic columns；
Mobile phase：0.1% aqueous formic acid of A phases, B phase acetonitriles；
Flow velocity：0.3mL/min；
Column temperature：40℃；
Gradient elution：
Mass Spectrometry Conditions：Capillary voltage 3.5kv, orifice potential 35kv, 500 DEG C of desolventizing temperature, desolventizing gas flow 800L/
Min, cone hole backflow airflow amount 30L/hr collide chamber pressure 3.0 × 10-3Mbar, low side resolution ratio 1:2.5V, high-end resolution ratio 1:
15.0V, ion energy 1:0.5, low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V, ion energy 2:1.0, ion source temperature
Degree：150 DEG C, extractor pressure：3.0V, entrance lens voltage：0.5V collides gradient：1.0；
The MS detection parameters：
5) qualitative detection：As parent ion [M+2H]2+540.9 existed simultaneously with daughter ion 595.3, daughter ion 319.3 and with it is special
When the appearance time of peptide fragment standard working solution is consistent, then judge to judge to contain lactoferrin in sample to be tested；When not detecting simultaneously
To parent ion [M+2H]2+540.9 with daughter ion 595.3, daughter ion 319.3 when, then judge to judge without newborn iron in sample to be tested
Albumen；
6) quantitative detection：According to liquid chromatogram, the mass spectrogram of special peptide fragment standard working solution, with special peptide fragment quota ion
Peak area is a concentration of ordinate of abscissa, special peptide fragment standard working solution, draws special peptide fragment standard curve；
Liquid chromatogram, the mass spectrogram that test solution is handled according to lactoferrin standard working solution, with special peptide fragment quota ion pair
Chromatographic peak area and the special peptide fragment standard curve obtain special peptide fragment concentration in lactoferrin standard working solution processing test solution；
According to special peptide fragment concentration, root in the concentration of lactoferrin standard working solution, lactoferrin standard working solution processing test solution
Lactoferrin, which is calculated, according to following formula digests coefficient：
CLactoferrin=K × CSpecial peptide fragment, wherein K is enzymolysis coefficient；
The Degree of Enzymatic Hydrolysis that lactoferrin in coefficient correction sample to be tested test liquid is digested with lactoferrin, using the special peptide fragment
Standard curve calculates the content of lactoferrin in sample to be tested.
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| CN117347531B (en) * | 2023-12-05 | 2024-02-13 | 天津医科大学总医院 | Method for detecting protein by adopting double internal calibration quantity |
| CN117744892A (en) * | 2024-02-08 | 2024-03-22 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating heat treatment strength of pasteurization process line and application |
| CN117744892B (en) * | 2024-02-08 | 2024-05-07 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating heat treatment strength of pasteurization process line and application |
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