CN106770866A - It is capable of achieving the kit and assay method of human serum albumins identification and absolute quantitation - Google Patents
It is capable of achieving the kit and assay method of human serum albumins identification and absolute quantitation Download PDFInfo
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- CN106770866A CN106770866A CN201611013854.9A CN201611013854A CN106770866A CN 106770866 A CN106770866 A CN 106770866A CN 201611013854 A CN201611013854 A CN 201611013854A CN 106770866 A CN106770866 A CN 106770866A
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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Abstract
The kit and assay method of human serum albumins identification and absolute quantitation are capable of achieving, the kit is made up of standard substance and reaction reagent two parts, and standard substance includes：Feature peptide fragment and its internal standard peptide fragment；Reaction reagent includes：Ammonium hydrogen carbonate, dithiothreitol (DTT), iodoacetamide, trypsase, formic acid.Mix by certain volume ratio with reaction reagent by by sample containing human serum albumins, after incubating vibration, it is allowed to enzyme digestion reaction, then standard substance and/or final enzymolysis product is injected separately into high performance liquid chromatography tandem mass spectrum instrument, according to parent ion and the mass-to-charge ratio of daughter ion（m/z）And the retention time of feature peptide fragment（tR）Qualitive test is carried out, the absolute content for calculating human serum albumins is changed by chromatographic peak area.
Description
Technical field
The present invention relates to a kind of achievable human serum albumins identification and the detection kit and assay method of absolute quantitation,
Belong to inspection determination techniques field.
Background technology
Human serum albumins is, containing 585 single chain protein matter of amino acid residue, to account for the 40~60% of Total plasma protein,
It is main carriers in blood plasma, rises and maintain osmotic pressure, pH bufferings, carrier and trophism, the material of many poorly water-solubles can leads to
Cross in connection and transported, e.g., bilirubin, LCFA, bile salt, prostaglandin, steroid hormone, metal ion
And medicine etc..At present, the main method of detection human serum albumins has immune double diffusion method, IE, double contractings both at home and abroad
Urine method, the wolframic acid precipitation method and capillary electrophoresis etc..These methods have the shortcomings that easily pollution, background value are high, often make detection sensitive
Degree is reduced.Retrieval finds that the patent application of Application No. 201010134413.0 discloses human seralbumin in a kind of detection human milk
The method of protein content, though the high voltage capillary electrophoresis method of use has high accuracy, is often vulnerable to molecular size range and approaches
Albumen interference, make detection specificity, sensitivity decrease, and detection time is more long, uncomfortable isotopism/micro human serum albumins
Detection.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of human serum albumins detection kit, provide a kind of using this
The method that kit can overcome the identification and assay of the human serum albumins of prior art shortcoming.The kit gives
The standard substance for detecting human serum albumins and the reaction reagent that enzyme digestion reaction is carried out to sample containing seralbumin.Can be by standard
Human seralbumin is carried out in material and/or enzyme digestion reaction product (or containing the internal standard material) injection (super) High Performance Liquid Chromatography/Mass Spectrometry instrument
Determining the protein quantity.The invention provides a kind of specificity it is strong, precision is high, result accurately and reliably, it is easy to operate, can be in clinic
The upper a kind of detection kit and detection method for being used for human serum albumins assay in sample.
Technical scheme：
A kind of achievable human serum albumins identification and the kit of absolute quantitation, it is characterised in that：Including standard substance：
Standard substance includes feature peptide fragment and its internal standard peptide fragment, and peptide section sequence is：
The kit also includes reaction reagent：
Reaction reagent, consists of the following composition
The standard substance feature peptide fragment can be used alone, or be made into two or three composite character with other feature peptide fragments
Peptide fragment is used.Internal standard peptide fragment is made into two or more and mixes internal standard substance and uses with other internal standard peptide fragments.
The internal standard peptide fragment of the standard substance is that C, H, O, N on any in feature peptide fragment, two or multiple amino acid are same
Peptide fragment after the element mark of position, wherein, tetra- elements of C, H, O, N on an amino acid can be labeled simultaneously, or any 1~3
Element is labeled.Internal standard peptide fragment can be used alone, and also can be made into two or three with other feature peptide fragments and mix internal standard substance makes
With.
The internal standard substance that the standard substance feature peptide fragment and its internal standard peptide fragment are made into, can be single internal standard substance, double
Mixing internal standard substance or three mixing internal standard substances, can be used it is therein it is a kind of, two or three.
1) standard substance
1. the standard substance for being used to realize kit of the present invention is feature peptide fragment, is single standard material, or with pairs mixed
Standardization material or polyhybird standard substance, use one or more therein.
Single standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single standard material II
The ETYGEMADCCAK of feature peptide fragment 2
Single standard material III
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Double hybrid standard material II
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material III
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
Three hybrid standard materials
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
2. the internal standard peptide fragment for being used to realize kit of the present invention is single internal standard peptide fragment, or is made into polyhybird internal standard peptide fragment,
Using it is therein it is a kind of, two or more；
Single internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, be single internal standard substance, double mixing internal standard substances or
Polyhybird internal standard substance, using it is therein it is a kind of, two or more；
Single internal standard substance I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard substance II
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard substance III
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Single internal standard substance IV
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance IX
Double mixing internal standard substance I
Double mixing internal standard substance II
Double mixing internal standard substance III
Double mixing internal standard substance IV
Double mixing internal standard substance V
Double mixing internal standard substance VI
Three mixing internal standard substances
4. it is single dose or double to be used to the standard substance in the human serum albumins detection kit for realize the inventive method
Agent or multi-agent, according to above method independent assortment or select it is therein it is a kind of, two or more.
The standard substance is made into single dose, double agent or multi-agent.
The trypsase is at least one in sequence-level trypsase, trypsase.
The reaction reagent is made into single dose.
The human seralbumin egg implemented using the kit of above-mentioned achievable human serum albumins identification and absolute quantitation
White content assaying method, it is characterised in that：The method step is as follows：
(1) sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol (DTT), incubate vibration, add iodoacetamide, temperature
Vibration is educated, is placed to room temperature, add trypsase, incubate vibration, be eventually adding formic acid terminating reaction.
(2) final enzymolysis product in standard substance and (1) step is injected separately into high performance liquid chromatography-tandem mass instrument
Final enzymolysis product (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into high performance liquid chromatography-series connection by detection
Detected in mass spectrograph, by parent ion and the mass-to-charge ratio (m/z) and the retention time (t of feature peptide fragment of daughter ion after detectionR) enter
The absolute content of human serum albumins is calculated in row Qualitive test, chromatographic peak peak area change.
Temperature control in 25~60 DEG C of scopes, incubative time control in 0.5~12h, frequency of oscillation control 800~
2500rmp, vortex time control is controlled in 18-22 DEG C of scope in 2~10min, room temperature.
Trypsase and the control of sample total protein concentration ratio are 1/1 to 1/100.
The step (2) is：Final enzymolysis product in feature peptide fragment (one or two) and (1) step is injected separately into
Detected in (super) high performance liquid chromatography-tandem mass instrument, or by final enzymolysis product (containing the internal standard in internal standard substance and (1) step
Peptide fragment) detection in (super) high performance liquid chromatography-tandem mass instrument is injected separately into, by m/z and tRCarry out qualitative, chromatogram peak-to-peak face
The content of human serum albumins is calculated in product change.
Beneficial effect：The device have the advantages that being：The detection kit and detection method that the present invention is provided can be right
Human serum albumins carries out accurate sensitive qualitative and absolute quantitation in sample, with high specificity, sensitivity is high, result is accurate
Really, easy to operate the advantages of, can be used for the detection of human serum albumins in the clinically human tissue sample such as blood sample, urine sample.
The present invention includes standard substance (being made up of feature peptide fragment and its internal standard peptide fragment) and enzyme digestion reaction reagent；Using (super)
High performance liquid chromatography-tandem mass instrument is detected that this is that a class uses high performance liquid chromatography separation, tandem mass spectrum detection fragment
The equipment of ion, human serum albumins that can quickly, accurately, sensitively to micro-/trace carries out absolute quantitation.The method has behaviour
Make the characteristics of simplicity, high specificity, sensitivity and degree of accuracy high.
At present, the feature peptide fragment and its internal standard peptide fragment and enzyme digestion reaction examination for determining human serum albumins content are not yet developed
The kit of agent.The deficiency that successfully compensate for field of biological detection of the invention, can exactly to containing micro-/trace human seralbumin egg
White sample carries out absolute Qualitive test and absolute quantitation, with high specificity, easy to operate, sensitivity and high excellent of the degree of accuracy
Point.
Brief description of the drawings
Fig. 1 is characterized the second order mses figure (m/z=1245.9) of peptide fragment 1
Fig. 2 is characterized the second order mses figure (m/z=717.7) of peptide fragment 2
Fig. 3 is characterized the second order mses figure (m/z=812.3) of peptide fragment 3；
Specific embodiment
The present invention is further illustrated by the examples that follow, but claim of the invention is not limited only to embodiment.
The present invention includes human serum albumins detection kit and content assaying method two parts.
Human serum albumins detection kit is made up of standard substance and reaction reagent two parts：
1) standard substance
1. the standard substance for being used to realize kit of the present invention is feature peptide fragment, can be single standard material, or be made into
Double hybrid standard materials or polyhybird standard substance, can be used one or more therein.
Single standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single standard material II
The ETYGEMADCCAK of feature peptide fragment 2
Single standard material III
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Double hybrid standard material II
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material III
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
Three hybrid standard materials
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
2. it is used to realize internal standard peptide fragment (C, H, O or/and N quilt on any, two or multiple amino acid of kit of the present invention
Isotope marks), can be single internal standard peptide fragment, or be made into polyhybird internal standard peptide fragment, can be used it is therein it is a kind of, two kinds or many
Kind.
Single internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, hereinafter referred to as " internal standard substance ", is more beneficial for human blood
The qualitative and quantitative determination of pure albumen.Can be single internal standard substance, double mixing internal standard substances or polyhybird internal standard substance, can
Using it is therein it is a kind of, two or more.
Single internal standard substance I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard substance II
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard substance III
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Single internal standard substance IV
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance IX
Double mixing internal standard substance I
Double mixing internal standard substance II
Double mixing internal standard substance III
Double mixing internal standard substance IV
Double mixing internal standard substance V
Double mixing internal standard substance VI
Three mixing internal standard substances
4. be used to the standard substance in the human serum albumins detection kit for realize the inventive method can be single dose,
Can be double agent or multi-agent, can according to above method independent assortment or select it is therein it is a kind of, two or more.
(2) reaction reagent, the reaction reagent for being used to realize the inventive method is single dose, including：
The step of present invention determines human serum albumins content is as follows：
(1) sample is added into ammonium hydrogen carbonate, vibration is mixed, and adds dithiothreitol (DTT), incubates vibration, adds iodacetyl
Amine, incubates vibration, places to room temperature, adds trypsase, incubates vibration, is eventually adding formic acid terminating reaction.
(2) standard substance (or internal standard substance) and final enzymolysis product (or containing the internal standard material) are injected separately into (super) efficiently
Detected in liquid chromatography-tandem mass spectrometry instrument, by m/z and tRQualitive test is carried out, sample is calculated in the change of chromatographic peak peak area
The content of middle human serum albumins.
In 25~60 DEG C of scopes, incubative time is controlled in 0.5~12h, frequency of oscillation for usual step (1) heated culture temperature control
Control is in 800~2500rmp.Trypsase and the control of sample total protein concentration ratio are in 1/1 to 1/100, vortex time control
System is in 2~10min.
Feature peptide fragment (one or two) and final enzymolysis product are injected separately into (super) high-efficient liquid phase color by the step (2)
Detected in spectrum-tandem mass spectrometer, or internal standard substance and final enzymolysis product (containing the internal standard peptide fragment) are injected separately into (super) efficiently liquid
Detected in phase chromatogram-tandem mass spectrometer, by m/z and tRQualitive test is carried out, is changed by chromatographic peak peak area, using internal standard
Method or external standard method calculate the content of human serum albumins in sample.
Embodiment 1：
Sample：Human plasma
Double mixing internal standard substance I
Double mixing internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
(1) preparation of sample：Human plasma 0.2ml is taken, 1ml cell pyrolysis liquids are added, ultrasonically treated 5min, precision is drawn
400 μ L homogenates, add 1.6ml methyl alcohol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds double mixing internal standard peptide fragment I, plus
The μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (1000rmp) 5min, adds the μ of 100mmol/L dithiothreitol (DTT)s solution 400
L, 60 DEG C incubate vibration (1000rmp) 60min, place to after room temperature, adding 100mmol/L iodoacetamidos amine aqueous solution 400 μ L, 30
DEG C vibration (1000rmp) 30min is incubated, add the μ L of 2mg/ml trypsin solutions 200,50 DEG C incubate vibration (1000rmp)
60min, places to room temperature, adds the μ L of 10% aqueous formic acid 400, and terminating reaction, freeze-drying obtains final enzymolysis and produces
Thing.
(2) double mixing internal standard substance I and final enzymolysis product are injected separately into (super) high performance liquid chromatography-tandem mass instrument
Middle detection.The detection limit of feature peptide fragment 1 and 2 is 1ng, and relative standard deviation is less than 3.26%, and the rate of recovery is 96.1%~
101.1% (n=6).
Embodiment 2：
Sample：Human milk
Single internal standard substance I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard peptide fragment I：
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
1) preparation of sample：Human milk 1.0ml is taken, 1ml cell pyrolysis liquids are added, ultrasonically treated 5min, precision draws 400 μ L
Homogenate, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue is added.Internal standard peptide fragment I, adds 100mmol/
The μ L of L ammonium bicarbonate solns 800, be vortexed (1200rmp) 2min, adds the μ L of 150mmol/L dithiothreitol (DTT)s solution 300,60 DEG C of temperature
Vibration (1200rmp) 30min is educated, is placed to room temperature, add the μ L of 40mmol/L iodoacetamidos amine aqueous solution 500,30 DEG C of incubations to shake
(1200rmp) 40min is swung, the μ L of 5mg/ml trypsin solutions 200 are added, 55 DEG C of vibrations (1500rmp) incubate 45min, place
To room temperature, the μ L of 20% aqueous formic acid 250 are added, terminating reaction, freeze-drying obtains final enzymolysis product.
2) single internal standard substance I and final enzymolysis product are injected separately into (super) high performance liquid chromatography-tandem mass instrument
Detection, detection is limited to 1ng, and relative standard deviation is less than 3.14%, and the rate of recovery is 95.3%~100.8% (n=6).
Embodiment 3：
Sample：People's albuminuria
Double hybrid standard material II
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The DVFLGMFLYEYAR of feature peptide fragment 3
1) preparation of sample：People albuminuria 10ml is taken, is centrifuged, abandon or adopt supernatant, residue adds 1ml cell pyrolysis liquids, surpasses
Sound crushes 1min, and ice bath cracking 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, be centrifuged (2000rpm), abandons or adopts
Clear liquid, residue adds the μ L of 100mmol/L ammonium bicarbonate solns 1000, and be vortexed (1000rmp) 5min, adds the sulphur of 150mmol/L bis-
The μ L of threose alcoholic solution 100,50 DEG C incubate vibration (1200rmp) 25min, place to room temperature, add 40mmol/L iodoacetamidos
The μ L of amine aqueous solution 500,35 DEG C incubate vibration (1000rmp) 8h, add the μ L of 0.3mg/ml trypsin solutions 1000,35 DEG C of incubations to shake
(1500rmp) 12h is swung, is placed to room temperature, add the μ L of 20% aqueous formic acid 500, terminating reaction, freeze-drying is obtained most
Whole enzymolysis product.
2) double hybrid standard materials and final enzymolysis product are injected separately into (super) high performance liquid chromatography-tandem mass instrument
Detection, feature peptide fragment 1 is 1ng, and the detection limit 3ng of feature peptide fragment 3, relative standard deviation is less than 4.14%, and the rate of recovery is
95.7%~102.4% (n=6).
Embodiment 4：
Sample：Human milk
Three mixing internal standard substances
Three mixing internal standard peptide fragments：
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
1) preparation of sample：1ml human milks are taken, 1ml cell pyrolysis liquids, ultrasonically treated 5min, ice bath cracking 2h, precision is added
400 μ L homogenates are drawn, 1.6ml methyl alcohol is added, is centrifuged (2000rpm), abandon or adopt supernatant, residue is added.Three mixing internal standard peptides
Section, adds the μ L of 120mmol/L ammonium bicarbonate solns 700, and be vortexed (800rmp) 10min, adds 100mmol/L dithiothreitol (DTT)s molten
The μ L of liquid 300,50 DEG C incubate vibration (2000rmp) 3h, place to room temperature, add the μ L of 10mmol/L iodoacetamidos amine aqueous solution 500,
25 DEG C incubate vibration (2500rmp) 30min, add the μ L of 2mg/ml trypsin solutions 200, and 55 DEG C of vibrations (1500rmp) incubate
180min, places to room temperature, adds the μ L of 5% aqueous formic acid 500, and terminating reaction, freeze-drying obtains final enzymolysis and produces
Thing.
2) three mixing internal standard substances and final enzymolysis product are injected separately into (super) high performance liquid chromatography-tandem mass instrument
Detection, the detection limit of feature peptide fragment 1,2 and 3 is 2ng, and relative standard deviation is less than 4.25%, and the rate of recovery is 96.1~
101.7% (n=6).
Embodiment 5：
Sample：People's albuminuria
Single standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single standard material II
The ETYGEMADCCAK of feature peptide fragment 2
Single standard material III
The DVFLGMFLYEYAR of feature peptide fragment 3
Single internal standard peptide fragment III
The DVFLGMFLYEYAR of internal standard peptide fragment 3
1) preparation of sample：People albuminuria 10ml is taken, is centrifuged, abandon or adopt supernatant, residue adds 1ml cell pyrolysis liquids, surpasses
Sound crushes 1min, and ice bath cracking 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, be centrifuged (2000rpm), abandons or adopts
Clear liquid, residue adds independent internal standard peptide fragment II, adds the μ L of 150mmol/L ammonium bicarbonate solns 60, and be vortexed (1000rmp) 6min,
The μ L of 10mmol/L dithiothreitol (DTT)s solution 100 are added, 55 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add
The μ L of 50mmol/L iodoacetamidos amine aqueous solution 500,30 DEG C incubate vibration (1000rmp) 50min, add 2mg/ml trypsin solutions
400 μ L, 35 DEG C incubate vibration (1800rmp) 10h, place to room temperature, the addition μ L of 5% aqueous formic acid 500, terminating reaction,
Freeze-drying, obtains final enzymolysis product.
2) after single standard material I, II, III is mixed with single internal standard peptide fragment, it is injected separately into final enzymolysis product
Detected in (super) high performance liquid chromatography-tandem mass instrument, the detection of feature peptide fragment 3 is limited to 1ng, and relative standard deviation is less than
2.13%, the rate of recovery is 96.0%~102.2% (n=6), and feature peptide fragment 1 and 2 is used as evidence peptide fragment.
Embodiment 6：
Sample：Human serum
Double mixing internal standard substance V
Double mixing internal standard peptide fragment II
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The DVFLGMFLYEYAR of internal standard peptide fragment 3
1) preparation of sample：Human serum 0.2ml is taken, adds 1ml cell pyrolysis liquids, ultrasonically treated 5min, ice bath to crack 2h,
Precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds 50mmol/L carbon
The μ L of sour hydrogen ammonium salt solution 1600, be vortexed (1000rmp) 6min, adds the μ L of 10mmol/L dithiothreitol (DTT)s solution 100,55 DEG C of incubations to shake
(1200rmp) 30min is swung, is placed to room temperature, add the μ L of 50mmol/L iodoacetamidos amine aqueous solution 500,30 DEG C incubate vibration
(1000rmp) 50min, adds the μ L of 1mg/ml trypsin solutions 1000, and 55 DEG C incubate vibration (1800rmp) 3.5h, place extremely
After room temperature, the μ L of 5% aqueous formic acid 800 are added, terminating reaction, freeze-drying obtains final enzymolysis product.
2) double mixing internal standard substance V are injected separately into (super) high performance liquid chromatography-tandem mass instrument with final enzymolysis product
Middle detection, the detection of feature peptide fragment 1 and 3 is limited to 1ng, and relative standard deviation is less than 2.68%, and the rate of recovery is 95.0%~98.2%
(n=6), feature peptide fragment 2 is used as evidence peptide fragment.
Embodiment 6：
Sample：People's whole blood
Single internal standard substance VII
Single internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The DVFLGMFLYEYAR of internal standard peptide fragment 3
1) preparation of sample：0.5ml people's whole blood is taken, 1ml cell pyrolysis liquids are added, ultrasonically treated 15min, precision is drawn
1000 μ L homogenates, add 1.6ml methyl alcohol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds single internal standard peptide fragment I, II
And III, the μ L of 100mmol/L ammonium bicarbonate solns 1000 are added, be vortexed (800rmp) 5min, adds the sulphur threoses of 150mmol/L bis-
The μ L of alcoholic solution 500,60 DEG C incubate vibration (2000rmp) 3h, place to room temperature, add 100mmol/L iodoacetamido amine aqueous solutions
500 μ L, 35 DEG C incubate vibration (2500rmp) 30min, add the μ L of 2mg/ml trypsin solutions 400,55 DEG C of vibrations
(1500rmp) incubates 240min, places to room temperature, adds the μ L of 10% aqueous formic acid 900, and terminating reaction, freeze-drying is obtained
To final enzymolysis product.
2) after single internal standard substance is mixed with single internal standard peptide fragment I and III, it is injected separately into (super) with final enzymolysis product
Detected in high performance liquid chromatography-tandem mass instrument, the detection limit of feature peptide fragment 1,2 and 3 is 2ng, and relative standard deviation is less than
3.49%, the rate of recovery is 95.8~97.6% (n=6).
In above example：Standard substance feature peptide fragment is used alone or is made into two kinds, three kinds with other feature peptide fragments and mixes
Standardization substance migration.
The internal standard substance that standard substance feature peptide fragment is made into its internal standard peptide fragment, can be single internal standard substance, double mixing
Internal standard substance or three mixing internal standard substances, can be used it is therein it is a kind of, two or three.
Standard substance is made into single dose, double agent or multi-agent.
The trypsase is at least one in sequence-level trypsase, trypsase.The embodiment of above-mentioned replacement exists
Here do not repeat.
In a word, it is demonstrated experimentally that can be carried out to human serum albumins in sample completely using detection kit of the invention
Identification, and required absolute content measurement result is drawn, and high, the specific good, precision of sensitivity is high, do not receive inside and outside source
The pollution of material.
Detection kit of the invention and detection method have that specificity is strong, precision is high, result accurately and reliably, operation letter
Just the advantages of, can be used for human serum albumins assay in sample.
Protein sequence
Human serum albumins
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE
VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVPEVDVM
CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKC
ASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKE
CCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYK
TTLEKCCAAADPECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK
VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF
HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETFAEEGKKLVAASRAALGL
Human serum albumins
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF
AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVPEVDVMCTAF
HDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ
KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEK
PLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYKTTLE
KCCAAADPECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSK
CCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADI
CTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETFAEEGKKLVAASRAALGL
Claims (9)
1. the kit of a kind of achievable human serum albumins identification and absolute quantitation, it is characterised in that：Including standard substance：
Standard substance includes feature peptide fragment and its internal standard peptide fragment, and peptide section sequence is：
Combination is as follows：
(1), the standard substance feature peptide fragment is used alone, or is made into two or more composite character peptides with other feature peptide fragments
Section is used；
(2), internal standard peptide fragment is used alone, or is made into two or more with other feature peptide fragments and mixes internal standard substance and use；
(3), the internal standard substance that the standard substance feature peptide fragment and its internal standard peptide fragment are made into, is single internal standard substance, double mixing
Internal standard substance or three mixing internal standard substances, using it is therein it is a kind of, two or three；
(4), internal standard peptide fragment is made into two or more and mixes internal standard substance and uses with other internal standard peptide fragments.
2. the kit of achievable human serum albumins identification according to claim 1 and absolute quantitation, it is characterised in that：
The kit also includes reaction reagent：
Reaction reagent, consists of the following composition
3. the kit of human serum albumins identification and absolute quantitation can be realized according to claim 1, it is characterised in that：
1) standard substance
1. the standard substance for being used to realize kit of the present invention is feature peptide fragment, is single standard material, or with mixing mark in pairs
Quasi- material or polyhybird standard substance, use one or more therein.
Single standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single standard material II
The ETYGEMADCCAK of feature peptide fragment 2
Single standard material III
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Double hybrid standard material II
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The DVFLGMFLYEYAR of feature peptide fragment 3
Double hybrid standard material III
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
Three hybrid standard materials
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
The DVFLGMFLYEYAR of feature peptide fragment 3
2. the internal standard peptide fragment for being used to realize kit of the present invention is single internal standard peptide fragment, or is made into polyhybird internal standard peptide fragment, is used
It is therein it is a kind of, two or more；
Single internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of internal standard peptide fragment 2
The DVFLGMFLYEYAR of internal standard peptide fragment 3
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is single internal standard substance, double mixing internal standard substances or more mixed
Close internal standard substance, using it is therein it is a kind of, two or more；
Single internal standard substance I
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
Single internal standard substance II
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
Single internal standard substance III
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
Single internal standard substance IV
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
The ALVLIAFAQYLQQCPFEDHVK of internal standard peptide fragment 1
The ETYGEMADCCAK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The ETYGEMADCCAK of feature peptide fragment 2
The ETYGEMADCCAK of internal standard peptide fragment 2
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The DVFLGMFLYEYAR of feature peptide fragment 3
The DVFLGMFLYEYAR of internal standard peptide fragment 3
The ALVLIAFAQYLQQCPFEDHVK of feature peptide fragment 1
Single internal standard substance IX
Double mixing internal standard substance I
Double mixing internal standard substance II
Double mixing internal standard substance III
Double mixing internal standard substance IV
Double mixing internal standard substance V
Double mixing internal standard substance VI
Three mixing internal standard substances
4. be used to the standard substance in the human serum albumins detection kit for realize the inventive method be single dose or double agent or
Multi-agent, according to above method independent assortment or select it is therein it is a kind of, two or more.
4. the kit of human serum albumins identification and absolute quantitation can be realized according to claim 1, it is characterised in that：Institute
The internal standard peptide fragment for stating standard substance is after C, H, O, N on any in feature peptide fragment, two or multiple amino acid is isotopically labeled
Peptide fragment, wherein, tetra- elements of C, H, O, N on an amino acid can be labeled simultaneously, or any 1~3 element is labeled.
5. the kit of human serum albumins identification and absolute quantitation can be realized according to claim 1, it is characterised in that：Institute
State standard substance and be made into single dose, double agent or multi-agent.
6. people can realize the kit of human serum albumins identification and absolute quantitation according to claim 1, it is characterised in that：
The trypsase is at least one in sequence-level trypsase, trypsase.
7. the kit of human serum albumins identification and absolute quantitation can be realized according to claim 2, it is characterised in that：Institute
State reaction reagent and be made into single dose.
8. the human blood implemented using the achievable human serum albumins identification described in claim 1 and the kit of absolute quantitation
Pure determining the protein quantity method, it is characterised in that：The method step is as follows：
(1) sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol (DTT), incubate vibration, add iodoacetamide, incubation shakes
Swing, place to room temperature, add trypsase, incubate vibration, be eventually adding formic acid terminating reaction.
(2) final enzymolysis product in standard substance and (1) step is injected separately into high performance liquid chromatography-tandem mass instrument and is detected
Or final enzymolysis product (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into high performance liquid chromatography-tandem mass
Detected in instrument, by parent ion and the mass-to-charge ratio (m/z) and the retention time (t of feature peptide fragment of daughter ion after detectionR) determined
Property differentiate, the absolute content of human serum albumins is calculated in the change of chromatographic peak peak area.
Temperature control is controlled in 800~2500rmp in 25~60 DEG C of scopes, incubative time control in 0.5~12h, frequency of oscillation,
Vortex time control is controlled in 18-22 DEG C of scope in 2~10min, room temperature.
Trypsase and the control of sample total protein concentration ratio are 1/1 to 1/100.
9. human serum albumins content assaying method according to claim 8, it is characterised in that：The step (2) is：By spy
Final enzymolysis product is injected separately into (super) high performance liquid chromatography-tandem mass instrument in levying peptide fragment (one or two) and (1) step
Middle detection, or final enzymolysis product (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into (super) high-efficient liquid phase color
Detected in spectrum-tandem mass spectrometer, by m/z and tRCarry out it is qualitative, chromatographic peak peak area change calculate human serum albumins
Content.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015109543826 | 2015-12-17 | ||
| CN201510954382 | 2015-12-17 |
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| CN106770866A true CN106770866A (en) | 2017-05-31 |
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| CN201611013854.9A Pending CN106770866A (en) | 2015-12-17 | 2016-11-18 | It is capable of achieving the kit and assay method of human serum albumins identification and absolute quantitation |
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| CN108333265A (en) * | 2017-12-15 | 2018-07-27 | 东莞理工学院 | A kind of zebrafish dna methylase hypotype biological mass spectrometry relative quantitation method |
| CN112098540A (en) * | 2020-08-31 | 2020-12-18 | 首都医科大学附属北京朝阳医院 | Characteristic peptide fragment, detection method and kit |
| CN113219117A (en) * | 2021-05-27 | 2021-08-06 | 杭州广科安德生物科技有限公司 | Mass spectrometry method of TIMP1 protein standard substance |
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