CN102331453A - Method for detecting polypeptide in body fluid - Google Patents
Method for detecting polypeptide in body fluid Download PDFInfo
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- CN102331453A CN102331453A CN201110095778A CN201110095778A CN102331453A CN 102331453 A CN102331453 A CN 102331453A CN 201110095778 A CN201110095778 A CN 201110095778A CN 201110095778 A CN201110095778 A CN 201110095778A CN 102331453 A CN102331453 A CN 102331453A
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Abstract
The invention provides a method for detecting polypeptide in body fluid, which comprises the following steps of: adding the polypeptide with the known concentration and mass to charge ratio peak, which is used as an internal standard, into a body fluid sample; detecting the sample by a mass spectrometer; and detecting the concentration of the target polypeptide according to the internal standard. The invention also provides the internal standard for the method and a detection kit containing the internal standard. In the invention, due to the addition of the isotope labeling internal standard into the body fluid sample, in the process of carrying out MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) linear data acquisition, after the correction is carried out by an external standard, the correction is carried out again by the internal standard, so that each peak value in a peptides map is more accurate and the shifting of molecular weight is avoided. Meanwhile, each peak in the peptides map can be relatively quantified and the problem that the polypeptide in the body fluid is difficult to detect quantitatively is solved.
Description
Technical field
The present invention relates to the detection method of polypeptide, specifically a kind of method of utilizing mass spectrum that polypeptide in the body fluid is detected.
Background technology
Ground substance assistant laser desorption ionization flight time mass spectrum (Matrix-assisted laserdesorption lionization time of flight mass spectrometry; MALDI-TOF-MS) technology; It is one of proteomic techniques of develop rapidly in recent years; Have highly sensitive, accuracy is high and characteristics such as resolution height; For life science and clinical major disease early warning and auxiliary diagnosis etc. provide fast, high with the quantitative analysis means of testing, also be a kind of molecule labelling method of good screening disease simultaneously.
Present correlative study is the applied research of multi-dimensional chromatograph separation and electro-spray ionization source (ESI source) mass spectrophotometry coupling mostly; And for the mass spectrum of ground substance assistant laser desorption ionization (MALDI) source; Because the different poor reproducibility that cause experimental result of factors such as sample separation degree, the crystalline condition of sample on target and ionization efficiency of peptide segment have limited present technique in the breadboard widespread use of clinical medicine in each experiment.
Catherine Fens doctor Lau of University of Maryland points out in research report: " mass spectrum is a technology qualitatively, is not a quantitative technology." because no matter be ESI or MALDI ion gun, because the difference of molecular structure causes the mass spectral peak height and the protein content of getting along well that absolute relation is arranged, the protein molecular that isodose is different, the response on mass spectrum is different.
Up to the present, also there is not the effectively relevant report of detection by quantitative serum polypeptide technology.
Summary of the invention
The objective of the invention is to, a kind of method that detects polypeptide in the body fluid is provided to above-mentioned deficiency.
Another object of the present invention is to be provided for the interior mark of polypeptide in the above-mentioned detection body fluid.
It is in body fluid sample, to add the isotope labeling polypeptide at concentration known and mass-to-charge ratio peak as interior mark that the present invention detects the method for polypeptide in the body fluid, utilizes mass spectrometer to detect sample, according to the concentration of interior mark detection target polypeptides.
Said body fluid can be serum, urine or tissue fluid or the like.
Said mass spectrometer is preferably ground substance assistant laser desorption ionization flight time mass spectrum.
Although biological mass spectrometry has higher detection sensitivity, its detectability receives that abundant interfering material limits in the complex environment.For this reason, the inventive method comprises also that preferably employing SPE-C magnetic bead carries out enrichment to serum.
Mark can join in the sample after the SPE-C enrichment with magnetic bead is handled in above-mentioned, perhaps directly joins in the serum, is handling through the SPE-C enrichment with magnetic bead then.Before a kind of mode realizes simply more easily that with respect to a kind of mode in back and mark and hemepeptide are composed the peak and existed jointly in realizing more easily, and intensity is suitable.
In the in fact available dual mode of mark modify, isotope is modified or base group modification, under peptide and the situation that internal standard peptide overlaps, the interior mark that isotope is modified is to the qualitative of serum polypeptide and quantitatively play guiding acting in sample.It is also passable that internal standard peptide adds the group modification, but the factors of instability possibly occur after mark is beaten mass spectrum in the base group modification.Compare, isotope is modified and is stablized more, has therefore adopted isotope labeling in the method in the present invention.
Above-mentioned isotope labeling can be
13C or
15N or both combinations, isotope-labeled site can be any amino acid on the polypeptide.
Interior mark can be one or several through isotope-labeled polypeptide, specifically can adapt with polypeptide to be checked in the serum.
And then the present invention also provides a kind of interior mark that above-mentioned body fluid polypeptide detects that is used for, and it is made up of one or several isotope-labeled polypeptide, and the concentration of these polypeptide and mass-to-charge ratio peak are known.Preferred said polypeptide is the isotope labeling polypeptide of intrinsic polypeptide in the serum, and its mass-to-charge ratio peak position is in the noiseless zone of sample polypeptide finger-print to be checked.For example, its isotope labeling polypeptide that designs according to polypeptide to be checked.
Interior target design; Can adopt following mode to carry out: 1, plug hole formula; Promptly on existing polypeptide data basis, check that two ways does not have intrinsic polypeptide influence, then design the interior mark (the mark polypeptide is positioned at the noiseless zone of body fluid polypeptide finger-print promptly) between this molecular weight area.2, the peptide that itself exists in the hemepeptide, and identified successful peptide (promptly use polypeptide in the serum polypeptide after isotope labeling as interior mark).
In one embodiment of the invention, select to exist in the hemepeptide polypeptide NLGHGHKHERDQGHGHQ that has also identified as internal standard peptide, isotope labeling is at four straight chain amino acid G.
Further can also mark in above-mentioned be prepared into various detection kit, use with convenient.
The present invention marks through in body fluid sample, adding in the isotope labeling, and when MALDI-TOF line acquisition data, external standard is proofreaied and correct with interior mark after proofreading and correct once more, make peptide compose in each peak value more accurate, avoid the drift of molecular weight; Simultaneously, can carry out relative quantification, solve the problem that the body fluid polypeptide is difficult to detection by quantitative each peak in the peptide spectrum.
Description of drawings
Fig. 1 is the mass spectrogram after the standard items external standard is proofreaied and correct, and its mean molecular weight deviation is less than 100ppm;
Fig. 2 is a mark self check mass spectrogram in the Mr=1943.65Da;
Fig. 3 is that serum+interior mark detects mass spectrogram;
The relative intensity of Fig. 4 polypeptide 1943.65 and the relation of concentration.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
Experiment purpose:
Proofread and correct optimization system repeatability and stable to 1943.65 as interior mark.
Experiment material:
(1) vacuum test tube BD Vacutainer SST (' tiger-top ') tube (Becton Dickinsoncat.no.367988)
(2) polypropylene EP pipe (being applicable to-80 ℃)
(3) rifle head Eppendorf standard tips
(4) comb Eppendorf safe-lock
(5) sub-bottling or pipe Eppendorf safe-look, Biozym PCR tubes (#710950)
(6) solvent bottle Nalgene FEP (Teflon) bottles
(7)MTPs?Greiner?PP，natural，MTP
(8) 0.1%TFA (chromatographic grade)! Severe corrosive 1ulTFA joins in the 1000ul Milli-Q water, mixes
(9) 100% acetone Acetone (chromatographic grade) J.K.Baker LOT NO.E32E11
(10) 100% ethanol Ethanol (chromatographic grade) J.K.Baker LOT NO.E39B24
(11) 100% acetonitrile Acetonitrile
(12) 10mM ammonium acetate Ammonium acetate (chromatographic grade)
(13) the 77mg ammonium acetate is dissolved in the 100ml Milli-Q water
(14) Peptide Calibration Standard (external standard) Bruker Daltonik; Cat.no.206195; Protein Calibration Standard I (external standard) BrukerDaltonik; Cat.no.206355
(15) Human serum (human serum) (Sigma; Cat.no.S7023)
(16) magnetic bead SPE-C weak cation magnetic bead kit YiXin Industry (Beijing) Science and Technology Ltd.
(17)α-Cyano-4-hydroxycinnamic?acid(HCCA)Bruker?Daltonik；1g#201344，5g#203072
(18)AnchorChip?600um #209514
Instrument and equipment:
(1) hydro-extractor (Bioyong)
(2) pipettor (Eppendorf)
(3) magnet stand Robotic separation device for 96 microtiter flate formatMagnetseparator (Bruker Daltonik)
(4) mass spectrometer: autoflexTMII (Bruker Daltonik)
(5) vacuum spinner: labconco Centrivap Centrifugal Concentratots andCold Traps
(6) related software: data acquisition FlexControl 2.2; Software FlexAnalysis3.0 with the aid of pictures; Analysis software ClinProTools 2.1 (Bruker DALTONICS company)
Experiment condition:
(1) sample type: synthetic polypeptide 1943.65Da NLGHGHKHERDQGHGHQ
(2) mass spectrometer: microflex, German Bruker
(3) analysis software: FlexAnalysis3.0, German Bruker
(4) acquisition parameter: sampling laser is 10%～20%, and every 50shots adopts a figure and adds up altogether 4 and scheme 200shots, acquisition range 800-10000Da
Experimental procedure:
(1) vibrate up and down lentamente heparin tube 5 times makes the coagula mixing in the blood.Room temperature (25 ℃) was vertically placed 1 hour, made blood clotting.(blood must accurately condense 1 hour, otherwise sample difference setting time can cause the peptide spectrum different artificially.) under the room temperature, with clinical centrifuge with the centrifugal SST pipe of 1.400-2.000g 10 minutes.Draw supernatant (serum) in the sample hose of mark of correspondence.Frozen immediately blood serum sample is in-80 ℃ of refrigerators.Avoid multigelation.
(2) from 4 ℃ of refrigerators, take out SPE-CM bead suspension one pipe, be inverted repeatedly, make magnetic bead fully, be suspended in the liquid phase uniformly.In the 200ul sample hose, add 2ul SPE-CM magnetic bead suspension, 95ul SPE-CB, 5ul serum (step 1 serum) are inhaled repeatedly and are beaten for several times, and magnetic bead and SPE-CB, serum are mixed, and room temperature leaves standstill 5min.Sample hose is placed on the magnetic bead separation vessel, makes the adherent 1min of magnetic bead, magnetic bead and fluid separation applications, treat liquid clarification after, exhaustion liquid.Sample hose in the last step is removed from the magnetic bead separation vessel, and in above-mentioned sample hose, added 100ul SPE-CW, inhale repeatedly and beat for several times, magnetic bead and SPE-CW are mixed, room temperature leaves standstill 2min.Sample hose is transferred on the magnetic bead separation vessel, is made the adherent 1min of magnetic bead, magnetic bead and fluid separation applications, treat liquid clarification after, exhaustion liquid.Sample hose is removed from the magnetic bead separation vessel, and in above-mentioned sample hose, added 10ul SPE-CE (being made up of SPE-CE1, SPE-CE2), inhale repeatedly and make a call on 10 times, magnetic bead and SPE-CE are mixed, room temperature leaves standstill 5min, gets sample eluent.
The 0.4mg/ml HCCA matrix of (3) getting 5.0ul is in the 200ul sample hose; Mark in the sample eluent that adds 1.0ul and the 1.0ul 100fmol/ul (can be per sample, crystallization, humiture etc. suitably adjusting wash-out liquid measure); Inhale repeatedly to make a call to more than 10 times with the application of sample rifle and mix; Get the above-mentioned mixing material of 1ul, point is on the 600umAnchorchip sample position, and it is residual that room temperature is placed to dry no liquid.
Experimental result:
As shown in Figure 1, to proofread and correct through the standard items appearance, the mean molecular weight deviation is less than 100ppm.As shown in Figure 2, the specific charge of the interior mark self-detection result of 1943.65Da is 1943.55, conforms to basically with notional result.As shown in Figure 3, interior mark is added in the serum, it has formed a significant peak at 1943.5 places.
Experiment purpose and principle
Synthetic 1943.65 isotopic peak 1947.65, it is quantitative that mark carries out polypeptide in utilizing.
F=(As/ms)/(Ar/mr) wherein As and Ar is respectively internal standard compound and its isotopic peak intensity, and ms and mr are respectively internal standard compound and its isotopic amount of adding.Get the component solution sample introduction to be measured that contains internal standard compound under each kind item again; The record mass spectrogram; Again according to the component solution peak intensity to be measured that contains internal standard compound; Calculate content (mi): mi=f * Ai/ (As/ms), wherein Ai and As are respectively the peak intensity of test sample and internal standard compound, and ms is for adding the amount of internal standard compound.
Experimental procedure:
(1) vibrate up and down lentamente heparin tube 5 times makes the coagula mixing in the blood.Room temperature (25 ℃) was vertically placed 1 hour, made blood clotting.(blood must accurately condense 1 hour, otherwise sample difference setting time can cause the peptide spectrum different artificially.) under the room temperature, with clinical centrifuge with the centrifugal SST pipe of 1.400-2.000g 10 minutes.Draw supernatant (serum) in the sample hose of mark of correspondence.Frozen immediately blood serum sample is in-80 ℃ of refrigerators.Avoid multigelation.
(2) from 4 ℃ of refrigerators, take out SPE-CM bead suspension one pipe, be inverted repeatedly, make magnetic bead fully, be suspended in the liquid phase uniformly.In the 200ul sample hose, add 2ul SPE-CM magnetic bead suspension, 95ul SPE-CB, 5ul serum are inhaled repeatedly and are beaten for several times, and magnetic bead and SPE-CB, serum are mixed, and room temperature leaves standstill 5min.Sample hose is placed on the magnetic bead separation vessel, makes the adherent 1min of magnetic bead, magnetic bead and fluid separation applications, treat liquid clarification after, exhaustion liquid.Sample hose in the last step is removed from the magnetic bead separation vessel, and in above-mentioned sample hose, added 100ul SPE-CW, inhale repeatedly and beat for several times, magnetic bead and SPE-CW are mixed, room temperature leaves standstill 2min.Sample hose is transferred on the magnetic bead separation vessel, is made the adherent 1min of magnetic bead, magnetic bead and fluid separation applications, treat liquid clarification after, exhaustion liquid.Sample hose is removed from the magnetic bead separation vessel, and in above-mentioned sample hose, added 10ul SPE-CE (being made up of SPE-CE1, SPE-CE2), inhale repeatedly and make a call on 10 times, magnetic bead and SPE-CE are mixed, room temperature leaves standstill 5min, obtains sample eluent.
The 0.4mg/ml HCCA matrix of (3) getting 5.0ul is in the 200ul sample hose; Add the sample eluent of 1.0ul and the interior mark of the isotope polypeptide 1947.65 of 0.5ul 200fmol/ul; And 0.5ul50fmol/ul 1943.65 obtain sample 1, and according to said method respectively configuration add 1943.65 the sample 2～6 that 0.5ul concentration is 100fmol/ul, 200fmol/ul, 400fmol/ul and 800fmol/ul, 1600fmol/ul, inhale repeatedly to make a call to more than 10 times with the application of sample rifle and mix; Get the above-mentioned mixing material of 1ul; Point is on 600um Anchorchip sample position, and it is residual that room temperature is placed to dry no liquid, respectively sample 1～6 measured.
Experiment condition:
Available isotope labeling comprises C13/N15, and marker site can be any amino acid in the peptide sequence.Integrated cost and synthetic complexity, selection 15N serves as a mark and is prone to synthetic straight chain amino acid (G, L) is that the interpolation site is preferable.Among the NLGHGHKHERDQGHGHQ, 1 L site, 4 G sites is arranged, be elected to be 4 G sites and synthesize general peptide and isotope peptide.Finally, synthetic interior mark peptide sequence is NLGHGHKHERDQGHGHQ, adds the 15N isotope labeling respectively in 4 G sites, and m/z of mark polypeptide is 1943.65Da and 1947.65Da in this.
(1) sample type: synthetic polypeptide 1943.65Da and 1947.65Da
(2) mass spectrometer: microflex, German Bruker
(3) analysis software: FlexAnalysis3.0, German Bruker
(4) acquisition parameter: sampling laser is 10%～20%, and every 50shots adopts a figure and adds up altogether 4 and scheme 200shots, acquisition range 800-10000Da
Experimental result:
The result shows that 1943Da and 1947Da can detect respectively clearly, explains that sensitivity and resolution can reach interior target basic demand.As shown in Figure 4, can detect the polypeptide that is low to moderate 2pmol/ul through the inventive method.
Claims (10)
1. a method that detects polypeptide in the body fluid is characterized in that, the isotope labeling polypeptide that in body fluid sample, adds concentration known and mass-to-charge ratio peak utilizes mass spectrometer to detect sample as interior mark, detects the concentration of target polypeptides according to interior mark.
2. the method for claim 1 is characterized in that, wherein said body fluid is serum, urine or tissue fluid.
3. method as claimed in claim 2 is characterized in that, also comprises when said body fluid is serum adopting the SPE-C magnetic bead that serum is carried out enrichment.
4. the method for claim 1 is characterized in that, said mass spectrometer is a ground substance assistant laser desorption ionization flight time mass spectrum.
5. the method for claim 1 is characterized in that, said isotope labeling polypeptide is to add isotope labeling according to target polypeptides to be checked to obtain.
6. method as claimed in claim 5 is characterized in that said isotope labeling does
13C or
15N.
7. interior mark that is used for each said method of claim 1～6, it is the isotope labeling polypeptide at 1 or several concentration known and mass-to-charge ratio peak.
8. mark is characterized in that said polypeptide is the isotope labeling polypeptide of intrinsic polypeptide in the serum in as claimed in claim 7, and its mass-to-charge ratio peak position is in the noiseless zone of sample polypeptide finger-print to be checked.
9. like claim 7 or 8 described interior marks, it is characterized in that said isotope labeling does
13C or
15N.
10. contain each said interior target detection kit of claim 7～9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239423A (en) * | 2020-01-17 | 2020-06-05 | 杭州汇健科技有限公司 | Molecular weight correction standard kit for polypeptide or protein mass spectrometry detection and preparation method and use method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440121B (en) * | 2008-12-31 | 2010-10-13 | 马庆伟 | System for separating protein polypeptide in blood serum and reagent kit thereof |
| US20100261279A1 (en) * | 2009-04-14 | 2010-10-14 | Ranish Jeff | Mass spectrum-based identification and quantitation of proteins and peptides |
| CN101963595A (en) * | 2010-09-02 | 2011-02-02 | 毅新兴业（北京）科技有限公司 | Serum polypeptide internal standard correction detection method |
-
2011
- 2011-04-15 CN CN201110095778A patent/CN102331453A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440121B (en) * | 2008-12-31 | 2010-10-13 | 马庆伟 | System for separating protein polypeptide in blood serum and reagent kit thereof |
| US20100261279A1 (en) * | 2009-04-14 | 2010-10-14 | Ranish Jeff | Mass spectrum-based identification and quantitation of proteins and peptides |
| CN101963595A (en) * | 2010-09-02 | 2011-02-02 | 毅新兴业（北京）科技有限公司 | Serum polypeptide internal standard correction detection method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239423A (en) * | 2020-01-17 | 2020-06-05 | 杭州汇健科技有限公司 | Molecular weight correction standard kit for polypeptide or protein mass spectrometry detection and preparation method and use method thereof |
| CN111239423B (en) * | 2020-01-17 | 2023-11-03 | 杭州汇健科技有限公司 | Application of polypeptide or protein mass spectrum detection molecular weight correction standard kit |
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Inventor after: Zhang Xueji Inventor after: Ma Qingwei Inventor after: Li Yan Inventor after: Zhang Yan Inventor after: Zhuo Guoyin Inventor after: Zhang Haiyan Inventor after: Hou Lingling Inventor after: Zhao Yanmei Inventor after: Xing Shuangyan Inventor before: Ma Qingwei Inventor before: Li Yan Inventor before: Zhang Yan Inventor before: Zhuo Guoyin Inventor before: Zhang Haiyan Inventor before: Hou Lingling Inventor before: Zhao Yanmei Inventor before: Xing Shuangyan |
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