CN102336828A - Multiple myeloma specific protein and its special detection kit - Google Patents
Multiple myeloma specific protein and its special detection kit Download PDFInfo
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- CN102336828A CN102336828A CN2010102387947A CN201010238794A CN102336828A CN 102336828 A CN102336828 A CN 102336828A CN 2010102387947 A CN2010102387947 A CN 2010102387947A CN 201010238794 A CN201010238794 A CN 201010238794A CN 102336828 A CN102336828 A CN 102336828A
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Abstract
The invention discloses multiple myeloma specific glycosylation protein and a special detection kit. An amino acid sequence of the protein is the sequence 1 in a sequence table, the amino acid residue from 342nd site of the amino terminal of the sequence 1 is the glycosylation site, a sugra chain formation connected with the site is [Hex]5[HexNAc]5[Fuc][NeuNAc]. The kit comprises a concanavalin affinity chromatography kit, a SDS-PAGE electrophoresis kit, a kit for enriching and separating trypsase, proleaseK and glycopeptide and a mass spectrometer. The multiple myeloma can be diagnosed by using the glycosylation site and the sequence of the multiple myeloma specific glycosylation protein of the invention. By detecting whether the human serum/blood plasma to be measured contains the specific glycosylation protein or not, the multiple myeloma specific glycosylation protein can be rapidly diagnosed by the kit of the invention.
Description
Technical field
The present invention relates to a kind of multiple myeloma specificity glycosylated protein and dedicated test test kit thereof.
Background technology
Multiple myeloma is the common a kind of malignant tumour of blood system, shows as monoclonal plasma cell dyscrasia.Its sickness rate occupies the 2nd of hematologic malignancies sickness rate.Slow, the concealment of multiple myeloma onset, early stage clinical manifestation is not obvious, easily by mistaken diagnosis.Major part has been a middle and advanced stage during prescription on individual diagnosis.Therefore need effective, simple early diagnosis marker of research and diagnostic method thereof, to realize early discovery (Oncologist, 2007, the 12:664 of multiple myeloma; Journal of Clinical Oncology, 2005,23:6345).
BSA is a strand spheroprotein matter in the human serum, also is the highest protein of content in the serum, generally is non-glycosylated protein.It has particular structure and many important physical functions.Comprise: nutritive value; Keep the blood oncotic pressure; Remove radical; Can also combine and transport many endogenous and exogenous material, as: serum Ca
2+, unconjugated bilirubin, free fatty acids and Triiodothyronine etc.The carrier of human serum BSA itself or many endogenous material and exogenous medicine after medicine and the albumin bound, can reduce its bioavailability and be increased in (Clin Perinatol, 2004,31:475 of intravital transformation period simultaneously; Journal of Infusion Nursing, 2006,29:260).BSA can influence its protein conformation through the increase that changes its state of oxidation such as glycosylation, disulfide linkage, so the joint efficiency of influence and medicine (British Journal of Pharmacology, 2007,151:580).In the clinical examination and diagnosis of multiple myeloma, the glycosylation albumin level has much relations with PD, is an important prognostic indicator.
Human serum BSA three-dimensional structure comprises similar domain: I (1-195) on three structures, II (196-383) and III (384-585), and each structural domain is divided into the subdomain of two similar spirane structures again.Connection between subdomain IA-IB, IIA-IIB, IIIA-IIIB then is respectively
106K extremely
119E,
292E extremely
315V,
492E extremely
511Three sections of A are stretched peptide chain, and each structural domain has different binding characteristics.Generally acknowledge the binding site that has two aromatic series or heterocyclic material at IIA and IIIA at present, have binding site (Journal of Biological Chemistry, 1999, the 274:29303 of longer chain fatty acid at IB and IIIB; Protein Engineering, 1999,12:439).The overwhelming majority medicines all combines with albuminous subdomain IIA, in this substructure zone the existence one can with the bigger hydrophobic region of many medicine bonded, other subdomain also possibly exert an influence to its combination.
Summary of the invention
The purpose of this invention is to provide a kind of multiple myeloma specificity glycosylated protein and dedicated test test kit thereof.
Multiple myeloma specificity glycosylated protein provided by the present invention, its aminoacid sequence are sequence 1 in the sequence table, and aminoterminal the 342nd amino acids residue of said sequence 1 is a glycosylation site, and the sugar chain that this site connects consists of [Hex]
5[HexNAc]
5[Fuc] [NeuNAc].
Above-mentioned multiple myeloma specificity glycosylated protein glycosylation site does
342N (N is a l-asparagine) belongs to the complexity sugar chain that N-connects, and sugar chain consists of [Hex]
5[HexNAc]
5[Fuc] [NeuNAc].Wherein, Hex representes hexose, and HexNAc representes the N-acetylaminohexose, and Fuc representes Fucose, and NeuNAc representes sialyl.
342The N glycosylation site is positioned at IIB, with the sero-abluminous difference of healthy subjects be from the 344th L-Ala of aminoterminal (
344A) sport Threonine (
344T), the caused glycosylation of this site mutation maybe be relevant with the generation of multiple myeloma.
The test kit of detection multiple myeloma specificity glycosylated protein provided by the present invention comprises ConA affinity chromatography test kit, SDS-PAGE electrophoresis kit, trypsinase, Proteinase K, glycopeptide concentration and separation test kit and mass spectrograph.
It is that 1: 1 the graphite carbon and the mixture of gac are the chromatography column of filler that said glycopeptide concentration and separation test kit comprises with mass ratio.The particle diameter scope of said graphite carbon is 5-8 μ m, and the activated carbon granule diameter range is 3-5 μ m.
Said ConA affinity chromatography test kit comprises ConA affinity column, binding buffer liquid and elution buffer; Said binding buffer liquid is by 20mM Tris-HCl, 0.15M NaCl, 1mM MnCl
2, 1mM CaCl
2Form the solution of pH7.4 with water; Said elution buffer is pH7.4, the aqueous solution of 0.5M methyl-α-D-glucopyranoside.
Said glycopeptide concentration and separation test kit also comprises sample-loading buffer, balance liquid and elutriant; Said sample-loading buffer and balance liquid are pH3.0, and concentration expressed in percentage by volume is 0.2% formic acid solution; Said elutriant is that to contain concentration expressed in percentage by volume be that the concentration expressed in percentage by volume of 0.2% formic acid is 30% acetonitrile solution.
The present invention also provides a kind of method that detects multiple myeloma specificity glycosylated protein, comprises the steps:
1) with the gp in the doubtful multiple myeloma patients serum of the method enrichment of ConA affinity chromatography, be contrast with healthy subjects serum;
2) the 10%SDS-PAGE electrophoretic separation 1) in obtain the gp in the multiple myeloma patients serum, downcut the gel contain discrepant protein band in 66kDa place and the healthy subjects serum;
3) will contain the gel of multiple myeloma specificity glycosylated protein, carry out the pancreatin in-gel digestion, and add the Proteinase K enzyme afterwards and cut, obtain double digestion solution;
4) with chromatographic separating process enriching step 3) in glycopeptide in the double digestion solution that obtains; The filler of the chromatography column that said chromatographic separating process is used is that mass ratio is 1: 1 the graphite carbon and the mixture of gac;
5) utilize substance assistant laser desorpted ionized mass spectroscopy glycopeptide, obtain the information of glycosylation site and sugar chain; If the proteic sequence that obtains is shown in sequence 1, and be glycosylation site from aminoterminal the 342nd amino acids, then this albumen is multiple myeloma specificity glycosylated protein.
The glycosylation site of multiple myeloma specificity glycosylated protein of the present invention capable of using and its sequence (with the sero-abluminous difference of healthy subjects be from the 344th L-Ala of aminoterminal (
344A) sport Threonine (
344T)) make a definite diagnosis multiple myeloma patients.Utilize test kit of the present invention, can through detecting whether contain the quick diagnosis of specificity glycosylated protein realization of the present invention in the human serum to be measured to multiple myeloma.
Description of drawings
Fig. 1 is the electrophorogram of sugar-protein in healthy subjects (NE) and three routine multiple myeloma patients (M1E, M2E and the M3E) serum after the ConA enrichment.
Fig. 2 is the electrophorogram of the seroglycoid of multiple myeloma patients (M1E) after the ConA enrichment, patients with lung cancer (L1E and L2E) and Pancreas cancer patients (P1E and P2E).
Fig. 3 is the substance assistant laser desorpted ionized flight time mass spectrum figure of multiple myeloma specificity glycosylated protein.
Fig. 4 is the glycopeptide mass spectrum of specificity glycosylated protein in the multiple myeloma patients.
Fig. 5 is that the glycopeptide structure of specificity glycosylated protein in the multiple myeloma patients is identified mass spectrum.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
The discovery of embodiment 1, multiple myeloma specificity glycosylated protein and structure are identified
One, the ConA affinity chromatography is to enrichment and the separation method and the effect evaluation thereof of gp in the serum
1, the preparation of ConA affinity column and chromatography enriching method
The ConA affinity column is available from Vector company.
The method of utilizing the ConA affinity column to carry out affinity chromatography enrichment gp is described below:
1) balance: with the binding buffer liquid of 10 times of volumes (by 20mM Tris-HCl, 0.15M NaCl, 1mM MnCl
2, 1mM CaCl
2Form the solution of pH7.4 with water) balance ConA affinity column;
2) go up appearance: 200 μ L serum and 1.8mL knot and damping fluid and PMSF (1mM/mL) mix, and 4 ℃ of mixings spend the night, then the ConA affinity column after the step 1) balance excessively;
3) flushing: with the unconjugated non-gp of binding buffer liquid flush away of 10 times of volumes;
4) wash-out: with elution buffer (pH7.4, the aqueous solution of 0.5M methyl-α-D-glucopyranoside) the wash-out gp of 5 times of volumes.
2, the ConA affinity chromatography is identified the enrichment and the isolating effect of gp in the serum
Contriver of the present invention has identified specificity and the susceptibility of ConA affinity chromatography to the enrichment of multiple myeloma specificity glycosylated protein through experiment, and concrete grammar is described below:
1) multiple myeloma associated glycoprotein susceptibility checking
In order to prove the susceptibility of 66kDa multiple myeloma associated glycoprotein, according to the method difference enrichment healthy subjects (NE) of step 1, the seroglycoid of three routine multiple myeloma patients (M1E, M2E and M3E).The gp that enrichment is obtained carries out the 10%SDS-PAGE electrophoretic separation.The result is as shown in Figure 1.Can know that from Fig. 1 three routine multiple myeloma patients seroglycoids have the protein band of a notable difference at the 66kDa place.Prove that this differential protein can be used as the multiple myeloma diagnosis marker.
2) specificity of multiple myeloma associated glycoprotein checking
In order to prove the specificity of ConA to 66kDa multiple myeloma associated glycoprotein, according to the method for step 1 respectively enrichment the seroglycoid of healthy subjects (NE), two routine patients with lung cancer (L1E and L2E), two routine Pancreas cancer patients (P1E and P2E) and a routine multiple myeloma patients (M1E).The gp that enrichment is obtained carries out the 10%SDS-PAGE electrophoretic separation.The result is as shown in Figure 2.Can know that from Fig. 2 the multiple myeloma patients seroglycoid is compared at the 66kDa place with healthy subjects, lung cancer and Pancreas cancer patients seroglycoid has a notable difference albumen, proves this differential protein specifically expressing in multiple myeloma patients.
Two, the evaluation of multiple myeloma specificity glycosylated protein
1. the evaluation of multiple myeloma specificity glycoprotein
After the repeatability and validity that have proved the ConA process of the test, the serum of healthy subjects and multiple myeloma patients has been carried out ConA glycosylated protein enrichment experiment according to the method for the step 1 in the step 1.The gp of healthy subjects serum (NE) and multiple myeloma patients serum and ConA enrichment is carried out the 10%SDS-PAGE electrophoresis.
The band of the 66kDa multiple myeloma associated glycoprotein (locating shown in the arrow among Fig. 2) that the multiple myeloma serum electrophoresis is obtained downcuts the gel that promptly obtains containing the multiple myeloma specific proteins, and this gel that contains the multiple myeloma specific proteins is carried out the pancreatin in-gel digestion.Concrete steps are:
1) gel that downcuts is placed 50% (concentration expressed in percentage by volume) acetonitrile solution decolouring that contains 25mM bicarbonate of ammonia after, place pure acetonitrile to dewater then, wait to do.
2) gel after will dewatering places 50 μ L to contain the 25mM ammonium bicarbonate soln of 10mM WR 34678, and 56 ℃, 1 hour; Place 50 μ L to contain the 25mM ammonium bicarbonate soln of 20mM iodo-acid amide, room temperature lucifuge 45 minutes then.Pure acetonitrile dehydration is waited to do.
3) add an amount of trypsin 5ng/ μ L according to the blob of viscose volume), 4 ℃ after 1 hour, 37 ℃ of enzymes are cut and are spent the night; Get supernatant.
The supernatant of above-mentioned enzyme being cut acquisition carries out substance assistant laser desorpted ionized flight time mass spectrum evaluation, and its mass spectrum is as shown in Figure 3.Utilize the MASCOT search engine retrieving result to be human serum albumin, the amino acid fraction of coverage is 52%.The aminoacid sequence of human serum albumin and with the mass spectrometric detection result of multiple myeloma specificity glycosylated protein to mate the peptide section as follows:
Match?to：ALBU_HUMAN?Score：200?Expect：2e-16
Serum?albumin?OS＝Homo?sapiens?GN＝ALB?PE＝1?SV＝2
Nominal?mass(M
r)：71317；Calculated?pI?value：5.92
NCBI?BLAST?search?of?
ALBU?HUMAN?against?nr
Unformatted?
sequence?string?for?pasting?into?other?applications
Taxonomy：
Homo?sapiens
Fixed?modifications：Carbamidomethyl(C)
Variable?modifications：Oxidation(M)
Cleavage?by?Trypsin：cuts?C-term?side?of?KR?unless?next?residue?is?P
Number?of?mass?values?searched：85
Number?of?mass?values?matched：31
Sequence?Coverage：52％
Matched?peptides?shown?in?Black：
1?MKWVTFISLL?FLFSSAYSRG?VFRRDAHKSE?VAHRFKDLGE?ENFKALVLIA
51?FAQYLQQCPF?EDHVKLVNEV?TEFAKTCVAD?ESAENCDKSL?HTLFGDKLCT
101?VATLRETYGE?MADCCAKQEP?ERNECFLQHK?DDNPNLPRLV?RPEVDVMCTA
151?FHDNEETFLK?KYLYEIARRH?PYFYAPELLF?FAKRYKAAFT?ECCQAADKAA
201?CLLPKLDELR?DEGKASSAKQ?RLKCASLQKF?GERAFKAWAV?ARLSQRFPKA
251?EFAEVSKLVT?DLTKVHTECC?HGDLLECADD?RADLAKYICE?NQDSISSKLK
301?ECCEKPLLEK?SHCIAEVEND?EMPADLPSLA?ADFVESKDVC?K
NYAEAKDVF
351?LGMFLYEYAR?RHPDYSVVLL?LRLAKTYETT?LEKCCAAADP?HECYAKVFDE
401?FKPLVEEPQN?LIKQNCELFE?QLGEYKFQNA?LLVRYTKKVP?QVSTPTLVEV
451?SRNLGKVGSK?CCKHPEAKRM?PCAEDYLSVV?LNQLCVLHEK?TPVSDRVTKC
501?CTESLVNRRP?CFSALEVDET?YVPKEFNAET?FTFHADICTL?SEKERQIKKQ
551?TALVELVKHK?PKATKEQLKA?VMDDFAAFVE?KCCKADDKET?CFAEEGKKLV
601?AASQAALGL。
2. multiple myeloma specificity glycoprotein double digestion
Get the supernatant of tryptic digestion, add Proteinase K, making its final concentration is 0.05 μ g/ μ L.37 ℃ of enzymes are cut and are spent the night, and promptly obtain the double digestion product.
3. multiple myeloma specificity glycoprotein glycopeptide concentration and separation
With graphite carbon granularity (particle diameter) scope is that 5-8 μ m and activated carbon granularity (particle diameter) scope are that 3-5 μ m mixes by 1: 1 (mass ratio); Be dissolved in and mass ratio blended graphite carbon and activated carbon mixture such as process in the acetonitrile solution of 80% (concentration expressed in percentage by volume), abundant mixing before the dress post.Carry out following glycopeptide concentration and separation process then:
1) dress post: get suitable amount of graphite carbon and activated carbon mixture solution, in the Tip that packs into the pipe, process the ZipTip post of length 5-8mm, and subsequent use with the acetonitrile solution flushing of 80% (concentration expressed in percentage by volume).
2) balance: add 30 μ L pH3.0, the formic acid solution of 0.2% (concentration expressed in percentage by volume), centrifugal 10 minutes of 30g repeats 3 times.
3) go up appearance: the sample that step 2 double digestion is good, using formic acid solution to transfer sample solution pH value is 3.0, uses pH3.0 then, the formic acid solution of 0.2% (concentration expressed in percentage by volume) is diluted to 30 μ L, is added in the good ZipTip post of balance centrifugal 10 minutes of 30g.
4) flushing: 20 μ L deionized waters, centrifugal 10 minutes of 30g repeats 2 times.
5) wash-out: 10 μ L pH4.0, contain 30% (concentration expressed in percentage by volume) acetonitrile solution of 0.2% (concentration expressed in percentage by volume) formic acid, centrifugal 5 minutes of 30g repeats 3 times, and collects, and promptly concentration and separation obtains the glycopeptide elute soln.
4. multiple myeloma specificity glycoprotein glycopeptide is identified
Carrying out substance assistant laser desorpted ionized flight time mass spectrum after getting 0.5 μ L glycopeptide elutriant and 0.5 μ L 20mg/mL PHB solution mixing identifies.Mass spectrum is shown in 4.
Utilize the MSMS technology, ion m/z 3169.2 is carried out the second order ms analysis, as shown in Figure 5.Resolve through NetNGlyc 1.0 Server prediction and artificial structure.Qualification result is: proteic glycosylation site does shown in the sequence 1
342NYT (" NYT " representes glycosylation site, and N is a l-asparagine, and Y is a tyrosine, and T is a Threonine) belongs to the complexity sugar chain that N-connects, and sugar chain consists of [Hex]
5[HexNAc]
5[Fuc] [NeuNAc].Wherein, Hex representes hexose, and HexNAc representes the N-acetylaminohexose, and Fuc representes Fucose, and NeuNAc representes sialyl.
342The NYT glycosylation site is positioned at IIB, with the sero-abluminous difference of healthy subjects be from the 344th L-Ala of aminoterminal (
344A) sport Threonine (
344T), the caused glycosylation of this site mutation maybe be relevant with the generation of multiple myeloma.
The above results shows that multiple myeloma specific proteins of the present invention and human serum albumin are only variant in a site, promptly
344A sports
344T, sequence is shown in sequence 1, and glycosylation site is a glycosylation site in the 342nd amino acids of sequence 1.
Utilize multiple myeloma specific proteins of the present invention glycosylation site (
342NYT) can the quick diagnosis multiple myeloma.Reclaiming sequence according to the method described above is the sequence shown in the sequence 1 in the sequence table, and carries out mass spectrometric detection, and whether detect from aminoterminal the 342nd amino acids is glycosylation site.If 342 sites are glycosylation sites, then point out this people possibly suffer from multiple myeloma.
Claims (7)
1. multiple myeloma specificity glycosylated protein, its aminoacid sequence is sequence 1 in the table, and aminoterminal the 342nd amino acids residue of said sequence 1 is a glycosylation site, and the sugar chain that this site connects consists of [Hex]
5[HexNAc]
5[Fuc] [NeuNAc], wherein, Hex representes hexose, and HexNAc representes the N-acetylaminohexose, and Fuc representes Fucose, and NeuNAc representes sialyl.
2. a test kit that detects multiple myeloma specificity glycosylated protein comprises ConA affinity chromatography test kit, SDS-PAGE electrophoresis kit, trypsinase, Proteinase K, glycopeptide concentration and separation test kit and mass spectrograph.
3. test kit according to claim 2 is characterized in that: it is that 1: 1 the graphite carbon and the mixture of gac are the chromatography column of filler that said glycopeptide concentration and separation test kit comprises with mass ratio.
4. test kit according to claim 3 is characterized in that: the particle diameter scope of said graphite carbon is 5-8 μ m, and the activated carbon granule diameter range is 3-5 μ m.
5. according to any described test kit among the claim 2-4, it is characterized in that: said ConA affinity chromatography test kit comprises ConA affinity column, binding buffer liquid and elution buffer; Said binding buffer liquid is by 20mM Tris-HCl, 0.15M NaCl, 1mM MnCl
2, 1mM CaCl
2Form the solution of pH7.4 with water; Said elution buffer is pH7.4, the aqueous solution of 0.5M methyl-α-D-glucopyranoside.
6. according to any described test kit among the claim 2-4, it is characterized in that: said glycopeptide concentration and separation test kit also comprises sample-loading buffer, balance liquid and elutriant; Said sample-loading buffer and balance liquid are pH3.0, and concentration expressed in percentage by volume is 0.2% formic acid solution; Said elutriant is that to contain concentration expressed in percentage by volume be that the concentration expressed in percentage by volume of 0.2% formic acid is 30% acetonitrile solution.
7. a method that detects multiple myeloma specificity glycosylated protein comprises the steps:
1) with the gp in the doubtful multiple myeloma patients serum of the method enrichment of ConA affinity chromatography, be contrast with healthy subjects serum;
2) the 10%SDS-PAGE electrophoretic separation 1) in obtain the gp in the multiple myeloma patients serum, downcut the gel contain discrepant protein band in 66kDa place and the healthy subjects serum;
3) with step 2) gel that downcuts, carry out the pancreatin in-gel digestion, add the Proteinase K enzyme afterwards and cut, obtain double digestion solution;
4) with chromatographic separating process enriching step 3) in glycopeptide in the double digestion solution that obtains; The filler of the chromatography column that said chromatographic separating process is used is that mass ratio is 1: 1 the graphite carbon and the mixture of gac;
5) utilize substance assistant laser desorpted ionized mass spectroscopy glycopeptide, obtain the information of glycosylation site and sugar chain; If the proteic sequence that obtains is shown in sequence 1, the 342nd amino acids is a glycosylation site, and then this albumen is multiple myeloma specificity glycosylated protein.
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| CN105651853A (en) * | 2016-01-21 | 2016-06-08 | 江南大学 | Subcellular structure characteristic N-linked carbohydrate chain and application thereof |
| JPWO2017043569A1 (en) * | 2015-09-08 | 2018-06-28 | Ｊｃｒファーマ株式会社 | A novel human serum albumin variant |
| CN110215740A (en) * | 2019-07-15 | 2019-09-10 | 大连医科大学 | A kind of preparation method of the hydrophilic pre-treatment silica gel material of amphoteric ion |
| CN113720900A (en) * | 2021-09-14 | 2021-11-30 | 首都医科大学附属北京朝阳医院 | Method for detecting M protein in serum based on MADLI-TOF MS technology |
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| CN101354379A (en) * | 2008-01-18 | 2009-01-28 | 许洋 | Reagent for detecting multiple myeloma characteristic protein by mass spectrum |
| CN101705302A (en) * | 2009-11-05 | 2010-05-12 | 北京大学人民医院 | Kit for assisting to diagnose multiple myeloma |
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| CN101354379A (en) * | 2008-01-18 | 2009-01-28 | 许洋 | Reagent for detecting multiple myeloma characteristic protein by mass spectrum |
| CN101705302A (en) * | 2009-11-05 | 2010-05-12 | 北京大学人民医院 | Kit for assisting to diagnose multiple myeloma |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPWO2017043569A1 (en) * | 2015-09-08 | 2018-06-28 | Ｊｃｒファーマ株式会社 | A novel human serum albumin variant |
| US11046751B2 (en) | 2015-09-08 | 2021-06-29 | Jcr Pharmaceuticals Co., Ltd. | Human serum albumin mutant |
| JP2021141893A (en) * | 2015-09-08 | 2021-09-24 | Ｊｃｒファーマ株式会社 | Novel human serum albumin mutant |
| JP7123216B2 (en) | 2015-09-08 | 2022-08-22 | Ｊｃｒファーマ株式会社 | A novel human serum albumin variant |
| US11634474B2 (en) | 2015-09-08 | 2023-04-25 | Jcr Pharmaceuticals Co., Ltd. | Human serum albumin mutant |
| JP7403595B2 (en) | 2015-09-08 | 2023-12-22 | Ｊｃｒファーマ株式会社 | Novel human serum albumin variants |
| CN105651853A (en) * | 2016-01-21 | 2016-06-08 | 江南大学 | Subcellular structure characteristic N-linked carbohydrate chain and application thereof |
| CN105651853B (en) * | 2016-01-21 | 2018-08-07 | 江南大学 | A kind of characteristic N- connection sugar chains of subcellular structure and its application |
| CN110215740A (en) * | 2019-07-15 | 2019-09-10 | 大连医科大学 | A kind of preparation method of the hydrophilic pre-treatment silica gel material of amphoteric ion |
| CN110215740B (en) * | 2019-07-15 | 2021-05-04 | 大连医科大学 | Preparation method of zwitter-ion hydrophilic pretreatment silica gel material |
| CN113720900A (en) * | 2021-09-14 | 2021-11-30 | 首都医科大学附属北京朝阳医院 | Method for detecting M protein in serum based on MADLI-TOF MS technology |
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