CN107202850A - The method of 5 kinds of low-abundance proteins in rich plasma or serum - Google Patents
The method of 5 kinds of low-abundance proteins in rich plasma or serum Download PDFInfo
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Abstract
The invention discloses the method for 5 kinds of low-abundance proteins in rich plasma or serum, it is post material using cellulose phosphate, chromatographic column running buffer includes 20 mM/ls of trishydroxymethylaminomethanes, 300 mM/ls of sodium chloride and 1 mM/l of ethylenediamine tetra-acetic acid, eluent includes 20 mM/ls of trishydroxymethylaminomethanes, 1 mol/L sodium chloride and 1 mM/l of ethylenediamine tetra-acetic acid, is enriched with while realization to 5 kinds of low-abundance proteins.Sample after enrichment is detected using high performance liquid chromatography joint high resolution mass spectrum, it is possible to identify above-mentioned 5 kinds of low-abundance proteins simultaneously carry out quantitative analysis.Substantially, method is easy for the concentration effect of the inventive method, it is not necessary to low cost using expensive equipment or consumptive material, is easy to be extended and applied；Antibody need not be used, the expense of detection can be substantially reduced, can also be avoided due to the uncertainty that antibody mass stability problem is brought.
Description
Technical field
The invention belongs to biological technical field, and in particular to the enrichment method of the low-abundance protein in blood plasma or serum.
Background technology
Proteomics can be studied protein with high throughput, available for finding a variety of disease diagnosis markers and medicine
Thing target spot.International human protein's group research tissue (Human Proteomic Organization, HUPO) was early in 2002
Start human plasma proteins' group plan (human plasma proteome project, HPPP), its scientific goal bag
Analysis human plasma and serum protein composition comprehensively are included, the difference degree of different ethnic group plasma proteins is determined, and determine not
With the individual difference under physiology and pathological state.However, plasma proteins abundance difference is huge, 21 kinds of main high abundances in blood plasma
And the albumen of median abundance, such as albumin, the antitrypsin of IgG, ɑ 1, the macroglobulin of ɑ 2, transferrins account for Total plasma protein
More than the 99% of content, and remaining 1% is made up of thousands of kinds of low abundance even extremely low-abundance protein.As leucocyte is situated between
Plain 6 usual concentration ranges are in 0~5ng/L, and albumin abundance is (35~50) × 109Ng/L, high low-abundance protein content can
Poor 1012, far beyond two dimensional electrophoresis (two-dimensional electrophoresis, 2DE) and other protein
The scope that omics technology can be determined.
When lesion occurs for human tissue organ, the albumen from pathological tissues or cell can enter hematological system, these
Pathology, the physiological status of protein and body are closely related, therefore, and it is to find and each that these protein, which are detected and analyzed,
One of most worthy sample of disease associated biomarkers is planted, these protein include many low-abundance proteins, and its albumen is dense
Degree is in ng/mL magnitudes or lower.We pay close attention to following 5 kinds of low-abundance proteins in the present invention, and this five kinds of albumen are dense in blood plasma
Degree is below 1 μ g/mL：
1. angiogenin (angiogenin, ANG)：It is a nuclease being made up of 123 amino acid, blood can be stimulated
Pipe is generated, and very important effect is played in tumor development, also closely related with nerve degenerative diseases.
2. myeloperoxidase (myeloperoxidase, MPO)：It is one of heme peroxidases superfamily member,
In the aniline blue particles for being primarily present in myeloid cell (mainly neutrophil leucocyte and monocyte).Myeloperoxidase
Gene pleiomorphism is relevant with the neurological susceptibility of a variety of diseases, and the rise of its expression is related to suffering from coronary artery disease neurological susceptibility,
The danger for suffering from myocardial infarction in early days can also be predicted.There is also correlation with lung cancer for myeloperoxidase.
3. lactoferrin (lactotranferrin, LTF)：It is a kind of multi-functional glycoprotein in siderophillin family,
It is primarily present in various juices.Lactoferrin has antibacterial and antiviral function, be innate immune system it is important into
Member, moreover it is possible to interacted with DNA, RNA and polysaccharide molecule, participate in many important physiology courses.There is research to think that lactoferrin is
The biomarker of potential oophoroma early diagnosis.
4. ALPHA-2u (lipocalin 2, LCN2)：It is the albumen played an important role in congenital immunity,
There is expression in neutrophil leucocyte, also there is low-level expression in kidney, prostate, respiratory tract and intestines and stomach.Damaged in acute kidney
When wound occurs, the LCN2 concentration in blood and urine can be raised rapidly, thus LCN2 can as acute injury of kidney biological marker
Thing.In addition, diagnosis marker can also be used as during chronic kidney disease, kidney transplant etc..
5. heparitinase (heparanase, HPSE)：Can be in cell surface or extracellular matrix by acetyl sulfate liver
Element is degraded to short chain polysaccharides.This albumen plays an important role in the growth, transfer and angiogenesis of tumour, thus has research
It is potential tumour medicine target to think heparitinase.In a variety of tumours such as breast cancer, oophoroma, in HPSE expression
Adjust, and be possible tumor markers with prognosis in the presence of necessarily associating.
In clinical examination practice, how the more difficult detection of these low-abundance proteins effectively detects and analyze low abundance egg
It is always the challenging problem in analysis science field in vain.
At present, for these low-abundance proteins quantitative analysis mainly by such as ELISA of the technology dependent on antibody etc. come
Realize, the accuracy of these methods is mainly determined by the quality of antibody, and antibody mass is easily affected by various factors, this
The unstable of measurement result can be caused.In addition all costly, when needing, detection simultaneously is more for these method prices based on antibody
Cost can be further up during individual antibody.
Mass-spectrometric technique achieves significant progress in recent years, can be with a variety of albumen of simultaneous quantitative, but with mass-spectrometric technique pair
Blood plasma or this kind of complex biological sample of serum carry out Direct Analysis there is also some shortcomings, influence this technology in clinical examination
Application.Difficult point is main at two aspects：On the one hand, the difference of blood plasma or the high and low abundance protein content in haemocyanin is huge
Greatly, if not further to blood plasma or blood serum sample handled and directly carry out conventional mass spectroscopy, the letter of low-abundance protein
Number easily covered by high-abundance proteins, cause low-abundance target protein not to be detected, low-abundance protein loss is high；Separately
On the one hand, the pre- of high-abundance proteins is rejected with methods such as multidimensional liquid chromatogram, electrophoresis pre-separations and by affine in immunity technology
Processing method and mass spectrometry, although the detection probability of low-abundance protein can be improved, but these methods are to instrument and equipment, behaviour
There is strict demand as technology, waste time and energy or need to use the consumptive material of costliness, be difficult to be promoted in actual applications.
Therefore, it is how simple and effective, inexpensively remove blood plasma or high-abundance proteins and rich plasma in serum or
Low-abundance protein in serum, is an important subject of proteomics, even more realizes effective inspection of low-abundance protein
The key surveyed and analyzed.
The content of the invention
In view of this, it is an object of the invention to provide the method for 5 kinds of low-abundance proteins in rich plasma or serum, without
Dependent on antibody, you can the simple and effective, high-abundance proteins that inexpensively remove in blood plasma or serum, so as to realize for blood
It is enriched with slurry or serum while 5 kinds of low-abundance proteins.After enrichment, high resolution mass spectrum pair can be combined using high performance liquid chromatography
This 5 kinds of low-abundance proteins are identified and quantitative analysis.
To achieve the above object, the present invention uses following technical scheme：
The method of 5 kinds of low-abundance proteins in rich plasma or serum, 5 kinds of low-abundance proteins are angiogenin, marrow
Peroxidase, lactoferrin, ALPHA-2u, heparitinase, comprise the following steps：
(1) post material is filled in centrifuge tube, and the solution of sodium hydroxide and sodium chloride is added to institute into the centrifuge tube
State post material and carry out alkali process, then hydrochloric acid solution is added into the centrifuge tube and acid treatment is carried out to the post material；Then, to described
Chromatographic column running buffer is added in centrifuge tube, supernatant is abandoned in centrifugation after suspend the post material and standing, repeatedly；Finally, to
The chromatographic column running buffer is added in the centrifuge tube, is attached to after the post material that suspends in void column, is obtained chromatographic column and seal
Close its outlet；
(2) according to the ratio of every 100 microlitres of blood plasma or serum 5 milliliters of chromatographic column running buffers of correspondence, the chromatogram is used
Post running buffer diluting plasma or serum, then loading is to the chromatographic column, after incubation, and opening the column outlet makes liquid
Face declines, when liquid level is down to the post material, adds the chromatographic column running buffer cleaning, and repeated washing is multiple；
(3) eluent is added into the chromatographic column, after incubation, opens the column outlet, collect elution fraction；
Wherein,
The post material is cellulose phosphate；
The chromatographic column running buffer includes 20 mM/ls of trishydroxymethylaminomethanes, 300 mM/ls of chlorinations
Sodium and 1 mM/l of ethylenediamine tetra-acetic acid, solvent is ultra-pure water, and pH value is 8.0；
The eluent includes 20 mM/ls of trishydroxymethylaminomethanes, 1 mol/L sodium chloride and 1 mM/l
Ethylenediamine tetra-acetic acid, solvent is ultra-pure water, and pH value is 8.0.
The elution fraction obtained by above-mentioned enrichment method, is detected using high performance liquid chromatography joint high resolution mass spectrum,
5 kinds of low-abundance proteins can be identified by data analysis, further, by being contrasted with not enriched testing result,
This it appears that：The mass spectrogram signal of 5 kinds of low-abundance proteins described in sample after enrichment is clean, no miscellaneous peak interference, peak face
Product is remarkably reinforced, and can directly be carried out relative quantitative assay, be illustrated concentration effect of the above method for 5 kinds of low-abundance proteins
Substantially.
In the specific embodiment of the present invention, in step (1), the alkali process is specific as follows：
1. 50 milliliters are added into 50 milliliters of centrifuge tubes equipped with 1 gram of phosphocellurose column material to rub/liter hydrogen-oxygen containing 0.05
Change the solution of sodium and 0.5 mol/L sodium chloride, rock and mixed, soak 5 minutes；
2. the centrifuge tube is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
3. 50 milliliters of ultra-pure waters are added to the centrifuge tube, suspend the post material, is put into centrifuge and centrifuges 1 point in 1000g
Supernatant pH value is measured after clock, supernatant is outwelled, if pH value is less than 10, terminates alkali process and is further processed, otherwise, weight
Multiple step is 3..
In the specific embodiment of the present invention, in step (1), the acid treatment is specific as follows：
4. 50 milliliter of 0.5 mol/L hydrochloric acid is added to the centrifuge tube equipped with post material after the alkali process, rocks progress
Mixing, soaks 5 minutes；
5. the centrifuge tube is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
6. 50 milliliters of ultra-pure waters are added to the centrifuge tube, suspension post material is put into centrifuge after 1000g is centrifuged 1 minute
Supernatant pH value is measured, supernatant is outwelled；If pH value is more than 4, terminates acid treatment and be further processed, otherwise repeat step
⑥。
In the specific embodiment of the present invention, in step (1), the time of the standing is 30 minutes.
In the specific embodiment of the present invention, in step (2), the time of the incubation is 30 minutes.
In the specific embodiment of the present invention, in step (3), the time of the incubation is 20 minutes.
In the specific embodiment of the present invention, the post material is cellulose phosphate powder.
Compared with prior art, the present invention has following beneficial technique effect：
The present invention utilizes cellulose phosphate, can be achieved for being enriched with blood plasma or serum while multiple low-abundance proteins.
Sample after enrichment is detected using liquid chromatogram joint mass spectrum, it is possible to identify this 5 kinds of low-abundance proteins simultaneously carry out relative quantification
Analysis.Significantly, method is easy for concentration effect of the present invention, it is not necessary to low cost using expensive equipment or consumptive material, is easy to promote
And application.In the enrichment method of the present invention and follow-up detection process, antibody need not be used, detection can be substantially reduced
Expense, also can remove the uncertainty brought due to antibody mass stability problem.
Brief description of the drawings
Figure 1A contrasts for the mass spectrogram of the plasma sample angiopoietin before and after being enriched with comparative example 1 and embodiment 1.
Figure 1B contrasts to be enriched with the mass spectrogram of lactoferrin in front and rear plasma sample in comparative example 1 and embodiment 1.
Fig. 1 C contrast to be enriched with the mass spectrogram of myeloperoxidase in front and rear plasma sample in comparative example 1 and embodiment 1.
Fig. 1 D are the mass spectrogram pair that ALPHA-2u in front and rear plasma sample is enriched with comparative example 1 and embodiment 1
Than.
Fig. 1 E contrast to be enriched with the mass spectrogram of heparitinase in front and rear plasma sample in comparative example 1 and embodiment 1.
Fig. 2A contrasts for the mass spectrogram of the blood serum sample angiopoietin before and after being enriched with comparative example 2 and embodiment 2.
Fig. 2 B contrast to be enriched with the mass spectrogram of lactoferrin in front and rear blood serum sample in comparative example 2 and embodiment 2.
Fig. 2 C contrast to be enriched with the mass spectrogram of myeloperoxidase in front and rear blood serum sample in comparative example 2 and embodiment 2.
Fig. 2 D are the mass spectrogram pair that ALPHA-2u in front and rear blood serum sample is enriched with comparative example 2 and embodiment 2
Than.
Fig. 2 E contrast to be enriched with the mass spectrogram of heparitinase in front and rear blood serum sample in comparative example 2 and embodiment 2.
Embodiment
In order to which the present invention is better described, technical scheme is readily appreciated, with reference to specific embodiment and attached
Figure, is described in further detail to the present invention.It should be understood that following embodiments is merely to illustrate the present invention, do not represent or
The scope of the present invention is limited, protection scope of the present invention is defined by claims.
Agents useful for same or the unreceipted production firm person of instrument, are the routine that can be commercially available in following examples
Product.
The enrichment of 5 kinds of low-abundance proteins in the blood plasma of embodiment 1
(1) chromatographic column is prepared
Cellulose phosphate powder needed for weighing is dispensed as post material (0.5 gram of post material/1 sample × sample number)
To multiple 50 milliliters of centrifuge tubes (equipped with 1 gram of post material in each centrifuge tube), and following processing is carried out respectively to each centrifuge tube：
1. added into centrifuge tube 50 milliliters it is molten containing 0.05 mol/L sodium hydroxide and 0.5 mol/L sodium chloride
Liquid, rocks and is mixed, and soaks 5 minutes；
2. the centrifuge tube that 1. step obtains is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
3. the centrifuge tube 2. obtained to step adds 50 milliliters of ultra-pure waters, and suspension post material is put into centrifuge and centrifuged in 1000g
Supernatant pH value is measured after 1 minute, supernatant is outwelled, if pH value is less than 10, step can be carried out 4., otherwise, repeat step is 3.；
4. the centrifuge tube 3. obtained to step adds 50 milliliter of 0.5 mol/L hydrochloric acid, rocks and is mixed, and soaks 5 points
Clock；
5. the centrifuge tube that 4. step obtains is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
6. the centrifuge tube 5. obtained to step adds 50 milliliters of ultra-pure waters, and suspension post material is put into centrifuge and centrifuged in 1000g
Supernatant pH value is measured after 1 minute, supernatant is outwelled；If pH value is more than 4, step can be carried out 7., otherwise repeat step is 6.；
7. 50 milliliters of chromatographic column running buffers are added in the centrifuge tube 6. obtained to step, again suspension post material, stood
After 30 minutes, it is put into centrifuge and is centrifuged 1 minute in 1000g, outwell supernatant；Repeat step is 7. twice；
Chromatographic column running buffer includes 20 mM/ls of trishydroxymethylaminomethanes, 300 mM/ls of sodium chloride and 1
MM/l ethylenediamine tetra-acetic acid, solvent is ultra-pure water, and pH value is 8.0；
8. standard is dispensed into after adding 4 milliliters of chromatographic column running buffers, suspension post material in the centrifuge tube 7. obtained to step
In void column got ready, dispensed per tubing string material to two void columns (the green skies Bioisystech Co., Ltd in Shanghai, specification is 6mL)
In, complete the dress post of chromatographic column.
(2) it is enriched with low-abundance target protein
According to the ratio of 5 milliliters of chromatographic column running buffer/100 microlitre blood plasma, with 10 milliliters of chromatographic column running buffers
200 microlitres of blood plasma are diluted, the chromatographic column (having closed outlet) equipped with post material that loading to step (1) is obtained is incubated 30 points
Zhong Hou, opening column outlet declines liquid level, when liquid level is down to post material, adds 5 milliliters of chromatographic column running buffer cleanings, altogether
Clean 6~8 times (totally 30~40 milliliters)；
4 milliliters of eluents are added, after being incubated 20 minutes, column outlet is opened, collects elution fraction, carry out next step point
Analysis or freezen protective；Eluent includes 20 mM/ls of trishydroxymethylaminomethanes, 1 mol/L sodium chloride and 1 mM/l
Ethylenediamine tetra-acetic acid, solvent is ultra-pure water, and pH value is 8.0.
Sample after enrichment (i.e. elution fraction) is detected using high performance liquid chromatography joint high resolution mass spectrum
(1) 500 μ L elution fractions samples are drawn to 10K ultra-filtration centrifuge tubes, 13000rpm/min is centrifuged 10 minutes, removes filter
Liquid is crossed, retains retention part；500 μ L elution fractions samples are drawn again to 10K ultra-filtration centrifuge tubes, are centrifuged 10 minutes, remove filtration
Liquid, retains retention part；It is repeated 4 times altogether, so that the concentration to amounting to 2mL elution fraction samples is completed, in 10K ultrafiltration centrifugation
The elution fraction sample concentrated in pipe；
(2) the 10K ultrafiltration centrifugation that 200 μ L concentration obtains for 50mmol/L ammonium bicarbonate soln to step (1) is drawn
Pipe, 13000rpm/min is centrifuged 10 minutes, removes filtered solution, retains retention part；
(3) dithiothreitol (DTT) is added in the 10K ultra-filtration centrifuge tubes obtained to step (2) so that final concentration of 20mmol/L,
60 DEG C are incubated 20 minutes；
(4) iodacetyl ammonia is added in the 10K ultra-filtration centrifuge tubes obtained to step (3) so that final concentration of 50mmol/L, room
Warm avoid light place 30 minutes；
(5) the 10K ultra-filtration centrifuge tubes that 200 μ L concentration obtain for 50mmol/L ammonium bicarbonate soln to step (4) are drawn,
13000rpm/min is centrifuged 10 minutes, removes filtered solution, retains retention part；It is repeated once；
(6) mass spectrum level pancreatin is added in the 10K ultra-filtration centrifuge tubes obtained to step (5), and (such as Promega produces
Trypsin gold) so that final concentration of 0.01 μ g/mL, and contain 0.4% (w/v) NaTDC (sodium
Deoxycholate, SDC), 37 DEG C are incubated 12~16 hours；
(7) the 10K super filter tubes that 100 μ L concentration obtain for 50mmol/L ammonium bicarbonate soln to step (6) are added,
13000rpm/min is centrifuged 10 minutes, collects the liquid of super filter tube retention；0.5~1% (v/v) trifluoroacetic acid is added to mix,
13000rpm/min is centrifuged 10 minutes, the liquid that absorption is retained to 1.5 milliliters of new centrifuge tubes；Use C18 suction nozzles (Thermo
Pierce article No.s 87784) after desalination, dried using instrument is concentrated in vacuo, it is molten with 0.1 (v/v) % formic acid weight, 13000rpm/min from
After the heart 10 minutes, supernatant fraction is transferred to mass spectrum loading pipe and detected；
Mass Spectrometer Method condition setting is as follows：
Use Q-exactive mass spectrographs and EASY-nLC 1000UPLC system.Liquid phase is using 60 minutes gradients, stream
Speed be 220nL/min, pre-column with AcclaimR PepMap100column (100 μm of ID, 2cm length, C18,5 μm,), analytical column with AcclaimR PepMap RSLC column (50 μm of ID, 15cm length, C18,2 μm,
).Mobile phase A is 0.1%FA (formic acid) aqueous solution, and Mobile phase B is 0.1%FA acetonitrile solutions.
Mass spectrometric data collection by Xcalibur 2.2SP1 softwares operate, one-level precursor scans scope be 350~
2000m/z, resolution ratio is 70000；15 polypeptide carries out fragmentation before selection signal ranking, and fragmentation mode is HCD, and energy is
27%.Carried out with Proteome Discoverer software (Version PD1.4, Thermo Scientific, USA)
Identification of Fusion Protein, Maxquant softwares (Version 1.4.0.8) carry out quantitative analysis.
Comparative example 1
(i.e. without chromatographic column and enrichment low-abundance protein is prepared the step of) is handled without enrichment, directly using efficient
Sample detection before liquid chromatogram combines high resolution mass spectrum to being enriched with, i.e.,：Using in 1 microlitre of blood plasma alternate embodiment 1 before enrichment
Concentration elution fraction sample (that is, the elution fraction sample of the concentration in detecting step obtained by (1)), use high performance liquid chromatography
The other conditions of joint high resolution mass spectrum detection are identical.
Mass Spectrometer Method result
(1) by Mass Spectrometer Method result this it appears that：After enrichment, a variety of high-abundance proteins concentration are significantly reduced, and are removed
Effect is obvious, for example：
SERPINA1 (accession number is P01009 albumen), the protein content after 100 microlitres of blood plasma enrichments is not enriched with 1 microlitre
Protein content ratio in blood plasma is 0.0761；ORM1 (accession number is P02763 albumen), the albumen after 100 microlitres of blood plasma enrichments
Amount is 0.0702 with the protein content ratio in 1 microlitre of non-rich plasma；A2M (accession number is P01023 albumen), 100 microlitres of blood
The protein content ratio in protein content and 1 microlitre of non-rich plasma after slurry enrichment is 0.0809；(accession number is P02787 egg to TF
In vain), the protein content ratio in the protein content and 1 microlitre of non-rich plasma after 100 microlitres of blood plasma enrichments is 0.0706；HP (is logged in
Number albumen for being P00738), the protein content after the enrichment of 100 microlitres of blood plasma is with the protein content ratio in 1 microlitre of non-rich plasma
0.1561；APOA2 (accession number is P02652 albumen), protein content and 1 microlitre of non-rich plasma after 100 microlitres of blood plasma enrichments
In protein content ratio be 0.3213；APOA1 (accession number is P02647 albumen), the protein content after 100 microlitres of blood plasma enrichments
It is 0.7349 with the protein content ratio in 1 microlitre of non-rich plasma；IGHA1 (accession number is P01876 albumen), 100 microlitres of blood
The protein content ratio in protein content and 1 microlitre of non-rich plasma after slurry enrichment is 0.2400；(accession number is P01859 to IGHG2
Albumen), the protein content ratio in protein content and 1 microlitre of non-rich plasma after the enrichment of 100 microlitres of blood plasma is 0.2775.Enrichment
The parent material volume of sample is 100 times of non-enriched sample, and the amount of these high-abundance proteins is not less than being enriched with sample after being enriched with
Product, illustrate that the ratio removed is more than 99%.
(2) by Mass Spectrometer Method result this it appears that：After enrichment, the concentration effect of 5 kinds of target low-abundance proteins is notable,
Refer to below for the analysis of mass spectrogram contrast and explanation before and after enrichment.
Figure 1A~1E, which respectively is, is enriched with front and rear plasma sample angiopoietin, breast in comparative example 1 and embodiment 1
Ferritin, myeloperoxidase, ALPHA-2u, the mass spectrogram contrast of heparitinase, wherein, Y-axis provides the phase of signal
To value, X-axis is the retention time of liquid chromatogram, and NL provides the absolute value (the 100% of correspondence relative value) at top in figure, Base
Peak m/z are the scopes of charge-mass ratio, and AA then provides peak area.Polypeptide in Figure 1A~1E corresponding to each base peak (base peak)
It is listed as follows：
Table 1：Polypeptide list corresponding to each base peak (base peak)
| Numbering | Polypeptide | M/z values | Position |
| SEQ ID NO：1 | YCESIMR | M/z=479.70971 | Figure 1A |
| SEQ ID NO：2 | ADAVTLDGGFIYEAGLAPYK | M/z=1036.02281 | Figure 1B |
| SEQ ID NO：3 | IGLDLPALNMQR | M/z=670.87142 | Fig. 1 C |
| SEQ ID NO：4 | SYPGLTSYLVR | M/z=628.33771 | Fig. 1 D |
| SEQ ID NO：5 | FLILLGSPK | M/z=494.31553 | Fig. 1 E |
As can be seen that having many on the mass spectrogram of the not enriched plasma sample angiopoietin of comparative example 1 from Figure 1A
Individual peak, including the Interference Peaks from high-abundance proteins, display signal are more miscellaneous；And blood plasma sample of the embodiment 1 after enrichment is handled
The mass spectrogram signal of product angiopoietin is clean, no miscellaneous peak, and NL values are obviously improved.
As can be seen that there is height on the mass spectrogram of lactoferrin in the not enriched plasma sample of comparative example 1 from Figure 1B
The Interference Peaks of abundance protein so that target protein information is blanked, and it is newborn in plasma sample of the embodiment 1 after enrichment is handled
The mass spectrogram signal of ferritin is clean, no miscellaneous peak, and NL values are high.
As can be seen that having in the not enriched plasma sample of comparative example 1 on the mass spectrogram of myeloperoxidase from Fig. 1 C
Multiple peaks, including the Interference Peaks from high-abundance proteins, display signal are more miscellaneous；And blood plasma of the embodiment 1 after enrichment is handled
The mass spectrogram signal of myeloperoxidase is clean in sample, no miscellaneous peak, and NL values are obviously improved.
As can be seen that having in the not enriched plasma sample of comparative example 1 on the mass spectrogram of ALPHA-2u from Fig. 1 D
Multiple peaks, including the Interference Peaks from high-abundance proteins, display signal are more miscellaneous；And blood plasma of the embodiment 1 after enrichment is handled
The mass spectrogram signal of ALPHA-2u is clean in sample, no miscellaneous peak, and NL values are obviously improved.
As can be seen that being not detected by the mass spectrum of heparitinase in the not enriched plasma sample of comparative example 1 from Fig. 1 E
Peak, thus it is speculated that for heparitinase abundance is low and is covered by high-abundance proteins signal, and blood plasma of the embodiment 1 after enrichment is handled
Heparitinase can be detected in sample, and NL values are high.
From above-mentioned Figure 1A~1E result, the signal in the mass spectrum of not enriched processing sample it is weak or be blanked or
Interference signal from high-abundance proteins is more miscellaneous, and signal is clean in the mass spectrum of sample after enrichment processing, and intensity is high, can carry out relative
Quantitative detection, illustrates that the above method has obvious concentration effect for this five kinds of albumen.
The enrichment of 5 kinds of low-abundance proteins in the serum of embodiment 2
With the plasma sample in blood serum sample alternate embodiment 1, other conditions are identical with embodiment 1, and sample is entered
Row enrichment.
Again in the same manner as in Example 1, using high performance liquid chromatography combine high resolution mass spectrum to sample after enrichment (i.e.
Elution fraction) detection.
Comparative example 2
Plasma sample in comparative example 1 is substituted with blood serum sample, other conditions are identical with comparative example 1, to not being enriched with
Blood serum sample detected.
Mass Spectrometer Method result
(1) by Mass Spectrometer Method result this it appears that：After enrichment, a variety of high-abundance proteins concentration are significantly reduced, and are removed
Effect is obvious.
(2) by Mass Spectrometer Method result this it appears that：After enrichment, the concentration effect of 5 kinds of target low-abundance proteins is notable,
Refer to below for the analysis of mass spectrogram contrast and explanation before and after enrichment.
Fig. 2A~2E, which respectively is, is enriched with front and rear blood serum sample angiopoietin, breast in comparative example 2 and embodiment 2
Ferritin, myeloperoxidase, ALPHA-2u, the mass spectrogram contrast of heparitinase, wherein, Y-axis provides the phase of signal
To value, X-axis is the retention time of liquid chromatogram, and NL provides the absolute value (the 100% of correspondence relative value) at top in figure, Base
Peak m/z are the scopes of charge-mass ratio, and AA then provides peak area.Polypeptide in Fig. 2A~2E corresponding to each base peak (base peak)
It is listed as follows：
Table 2：Polypeptide list corresponding to each base peak (base peak)
| Numbering | Polypeptide | M/z values | Position |
| SEQ ID NO：1 | YCESIMR | M/z=479.70971 | Fig. 2A |
| SEQ ID NO：2 | ADAVTLDGGFIYEAGLAPYK | M/z=1036.02281 | Fig. 2 B |
| SEQ ID NO：3 | IGLDLPALNMQR | M/z=670.87142 | Fig. 2 C |
| SEQ ID NO：4 | SYPGLTSYLVR | M/z=628.33771 | Fig. 2 D |
| SEQ ID NO：5 | FLILLGSPK | M/z=494.31553 | Fig. 2 E |
As can be seen that existing on the mass spectrogram of the not enriched blood serum sample angiopoietin of comparative example 2 from Fig. 2A
Interference Peaks, display signal is more miscellaneous；And the mass spectrogram signal of blood serum sample angiopoietin of the embodiment 2 after enrichment is handled
Totally, no miscellaneous peak, and NL values are obviously improved.
As can be seen that there is height on the mass spectrogram of lactoferrin in the not enriched blood serum sample of comparative example 2 from Fig. 2 B
The Interference Peaks of abundance protein so that target protein information is blanked, and it is newborn in blood serum sample of the embodiment 2 after enrichment is handled
The mass spectrogram signal of ferritin is clean, no miscellaneous peak, and NL values are high.
As can be seen that being deposited in the not enriched blood serum sample of comparative example 2 on the mass spectrogram of myeloperoxidase from Fig. 2 C
In the Interference Peaks of high-abundance proteins so that target protein information is blanked, and blood serum sample of the embodiment 2 after enrichment is handled
The mass spectrogram signal of middle myeloperoxidase is clean, no miscellaneous peak, and NL values are high.
As can be seen that being deposited in the not enriched blood serum sample of comparative example 2 on the mass spectrogram of ALPHA-2u from Fig. 2 D
In the Interference Peaks of high-abundance proteins so that target protein information is blanked, and blood serum sample of the embodiment 2 after enrichment is handled
The mass spectrogram signal of middle ALPHA-2u is clean, no miscellaneous peak, and NL values are high.
As can be seen that being not detected by the mass spectrum of heparitinase in the not enriched blood serum sample of comparative example 2 from Fig. 2 E
Peak, thus it is speculated that for heparitinase abundance is low and is covered by high-abundance proteins signal, and serum of the embodiment 2 after enrichment is handled
Heparitinase can be detected in sample.
From above-mentioned Fig. 2A~2E result, signal in the mass spectrum of not enriched processing sample is weak, is blanked or comes
Interference signal from high-abundance proteins is more miscellaneous, and signal is clean in the mass spectrum of sample after enrichment processing, and intensity is high, and it is relatively fixed to carry out
Amount detection, illustrates that the above method has obvious concentration effect for this five kinds of albumen.
Cellulose phosphate powder is as cation exchange column post material in above-described embodiment, and it can be purchased by commercial sources
Buy, such as Sigma-Aldrich, Spectrum Chemical companies of the U.S..
As can be seen here, the purpose of the present invention is achieved completely and effectively.The method and principle of the present invention is
Shown and illustrated in embodiment, in the case of without departing substantially from the principle, embodiment can make any modification.So,
Present invention comprises all variant embodiments based on claim spirit and right.
SEQUENCE LISTING
<110>Zhejiang Prov. Tumor Hospital
<120>The method of 5 kinds of low-abundance proteins in rich plasma or serum
<130> 2017
<160> 5
<170> PatentIn version 3.3
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Ala Pro Tyr Lys
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Ile Gly Leu Asp Leu Pro Ala Leu Asn Met Gln Arg
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Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg
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<213>People (homo sapiens)
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Claims (6)
1. the method for 5 kinds of low-abundance proteins in rich plasma or serum, 5 kinds of low-abundance proteins are angiogenin, marrow mistake
Oxide enzyme, lactoferrin, ALPHA-2u, heparitinase, comprise the following steps：
(1) post material is filled in centrifuge tube, and the solution of sodium hydroxide and sodium chloride is added to the post into the centrifuge tube
Material carries out alkali process, then adds hydrochloric acid solution to post material progress acid treatment into the centrifuge tube；Then, to the centrifugation
Chromatographic column running buffer is added in pipe, supernatant is abandoned in centrifugation after suspend the post material and standing, repeatedly；Finally, to described
The chromatographic column running buffer is added in centrifuge tube, is attached to after the post material that suspends in void column, is obtained chromatographic column and close it
Outlet；
(2) according to the ratio of every 100 microlitres of blood plasma or serum 5 milliliters of chromatographic column running buffers of correspondence, transported with the chromatographic column
Row buffering liquid diluting plasma or serum, then loading is to the chromatographic column, after incubation, and opening the column outlet makes under liquid level
Drop, when liquid level is down to the post material, adds the chromatographic column running buffer cleaning, repeated washing is multiple；
(3) eluent is added into the chromatographic column, after incubation, opens the column outlet, collect elution fraction；
Wherein,
The post material is cellulose phosphate；
The chromatographic column running buffer includes 20 mM/ls of trishydroxymethylaminomethanes, 300 mM/ls of sodium chloride and 1
MM/l ethylenediamine tetra-acetic acid, solvent is ultra-pure water, and pH value is 8.0；
The eluent includes 20 mM/ls of trishydroxymethylaminomethanes, 1 mol/L sodium chloride and 1 mM/l of second two
Amine tetraacethyl, solvent is ultra-pure water, and pH value is 8.0.
2. the method for 5 kinds of low-abundance proteins in rich plasma as claimed in claim 1 or serum, it is characterised in that step (1)
In, the alkali process is specific as follows：
1. 50 milliliters are added into 50 milliliters of centrifuge tubes equipped with 1 gram of phosphocellurose column material to rub/liter sodium hydroxide containing 0.05
With the solution of 0.5 mol/L sodium chloride, rock and mixed, soak 5 minutes；
2. the centrifuge tube is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
3. 50 milliliters of ultra-pure waters are added to the centrifuge tube, suspend the post material, is put into centrifuge after 1000g is centrifuged 1 minute
Supernatant pH value is measured, supernatant is outwelled, if pH value is less than 10, terminates alkali process and is further processed, otherwise, repeats to walk
Suddenly 3..
3. the method for 5 kinds of low-abundance proteins in rich plasma as claimed in claim 2 or serum, it is characterised in that step (1)
In, the acid treatment is specific as follows：
4. 50 milliliter of 0.5 mol/L hydrochloric acid is added to the centrifuge tube equipped with post material after the alkali process, rocks and mixed
Close, soak 5 minutes；
5. the centrifuge tube is put into centrifuge to centrifuge 1 minute in 1000g, outwells supernatant；
6. 50 milliliters of ultra-pure waters are added to the centrifuge tube, suspension post material is put into centrifuge and measured after 1000g is centrifuged 1 minute
Supernatant pH value, outwells supernatant；If pH value is more than 4, terminates acid treatment and be further processed, otherwise repeat step is 6..
4. such as the method for 5 kinds of low-abundance proteins in rich plasma according to any one of claims 1 to 3 or serum, its feature
It is, in step (1), the time of the standing is 30 minutes.
5. such as the method for 5 kinds of low-abundance proteins in rich plasma according to any one of claims 1 to 3 or serum, its feature
It is, in step (2), the time of the incubation is 30 minutes.
6. such as the method for 5 kinds of low-abundance proteins in rich plasma according to any one of claims 1 to 3 or serum, its feature
It is, in step (3), the time of the incubation is 20 minutes.
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