CN109633066A - A kind of inexpensive, simple and quick glycoprotein N- sugar chain analysis method - Google Patents
A kind of inexpensive, simple and quick glycoprotein N- sugar chain analysis method Download PDFInfo
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- CN109633066A CN109633066A CN201910025081.3A CN201910025081A CN109633066A CN 109633066 A CN109633066 A CN 109633066A CN 201910025081 A CN201910025081 A CN 201910025081A CN 109633066 A CN109633066 A CN 109633066A
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- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 21
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 21
- 238000004458 analytical method Methods 0.000 title abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 43
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims abstract description 16
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims abstract description 16
- 238000012545 processing Methods 0.000 claims abstract description 14
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 13
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 claims abstract description 9
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims abstract description 8
- 238000005932 reductive alkylation reaction Methods 0.000 claims abstract description 6
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 claims abstract description 5
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims abstract description 4
- 230000029087 digestion Effects 0.000 claims abstract description 4
- 238000000746 purification Methods 0.000 claims abstract description 4
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 claims abstract description 3
- 108010015899 Glycopeptides Proteins 0.000 claims abstract description 3
- 102000002068 Glycopeptides Human genes 0.000 claims abstract description 3
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- 239000000243 solution Substances 0.000 claims description 17
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
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- 238000012856 packing Methods 0.000 claims description 2
- 238000010183 spectrum analysis Methods 0.000 claims description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 2
- 101001055315 Homo sapiens Immunoglobulin heavy constant alpha 1 Proteins 0.000 description 9
- 102100026217 Immunoglobulin heavy constant alpha 1 Human genes 0.000 description 9
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- 238000004108 freeze drying Methods 0.000 description 6
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 5
- 101000749634 Homo sapiens Uromodulin Proteins 0.000 description 4
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- 238000001035 drying Methods 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 210000002700 urine Anatomy 0.000 description 4
- 102000005348 Neuraminidase Human genes 0.000 description 3
- 108010006232 Neuraminidase Proteins 0.000 description 3
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- 239000012535 impurity Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102400000058 Neuregulin-1 Human genes 0.000 description 1
- 108090000556 Neuregulin-1 Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- 238000013112 stability test Methods 0.000 description 1
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- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N2030/8836—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 saccharides
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of low costs, simple and quick glycoprotein N- sugar chain quantitative analysis method, it carries out reductive alkylation processing after including the following steps: (1) glycoprotein heat denatured；(2) the N- sugar chain on glycopeptide is removed using the digestion of PNGase F protein in super filter tube, N- sugar chain is collected by centrifugation；(3) hydrophilic Interaction Chromatography purification enrichment N- sugar chain is used；(4) after removing sialic acid, dry concentration；(5) Matrix-assisted laser desorption ionization analyzes N- sugar chain.Operation of the present invention is simple, quick, is widely portable to the analysis of the N- sugar chain of the glycoprotein of separate sources, analysis result is reliable and stable, and application prospect is wide.
Description
Technical field
The present invention relates to biochemical field more particularly to a kind of glycoprotein N- sugar chain analysis methods.
Background technique
The N- sugar chain connected on glycoprotein is by glucose, galactolipin, mannose, N- acetyl glucosamine, N- acetyl gala
The monosaccharide such as sugar, sialic acid pass through molecule made of glucosides key connection.N- sugar chain is catalyzed in endoplasm net surface through glycosyl transferase, with
Asparagine residue on newly-generated protein enters and one occurs in endoplasmic reticulum and golgiosome by N- glucosides key connection
Serial following process.N- glycosylation usually occurs in conservative amino acid sequence N-X-T/S (X ≠ P), according to outside pentasaccharides core
The extension system for enclosing sugar chain is different, can be divided into high mannose type, heterozygous and compound again, different sugar chain structures is each
The adjusting function important from performance.The experimental results show the invasion of cell surface N- sugar chain abnormal variation and tumour cell
With transfer etc. have substantial connection.The structure change of N- sugar chain is studied for illustrating work of the sugar chain in tumour and disease development
With the research and development of discovery and early diagnosis, the treatment or even drug of sugar chain marker are of great significance.
But N- sugar chain analysis difficulty is larger, essentially consists in its low content, type and structure is complicated, heterogeneity etc., therefore
It is (a kind of that the pre-treatment (purifying) of N- sugar chain quantitative analysis is particularly important the patent document that notification number is 102788720 B of CN
The full N- of glycoprotein connects sugar chain and its discrimination method in filter membrane auxiliary separation biological sample) disclose a kind of purifying of N- sugar chain and analysis
Method cuts N- sugar chain by PNGase F digestion, after obtaining N- sugar chain crude extract with 10KD molecular sieve filtration, uses
Sepharose 4B purifies N- sugar chain in centrifuge tube；Then by Matrix-assisted laser desorption ionization to it
It is analyzed.But the mass signal of the N- sugar chain finally generated is on the weak side, and the sugar chain type of identification is on the low side.
Summary of the invention
In order to enhance the Mass Spectrometer Method signal of N- sugar chain, present invention firstly provides a kind of low costs, simple and quick sugar
The pre-treating method of albumen N- sugar chain quantitative analysis, it includes the following steps:
(1) reductive alkylation processing is carried out after glycoprotein heat denatured；
(2) the N- sugar chain on glycopeptide is removed using the digestion of PNGase F protein in super filter tube, N- sugar chain is collected by centrifugation；
(3) hydrophilic Interaction Chromatography purification enrichment N- sugar chain is used；
(4) after removing sialic acid, dry concentration.
Pre-treating method above-mentioned, the processing of reductive alkylation described in step (1) are as follows: use dithiothreitol (DTT) and iodine respectively
Acetamide processing.
Further, the dithiothreitol (DTT) processing are as follows: 50-60 DEG C of oscillating reactions 30-60min.
Further, the iodoacetamide processing are as follows: final concentration 30-70mmol/L iodoacetamide is added and is protected from light instead in room temperature
Answer 0.5-1.5h.
Pre-treating method above-mentioned, endonuclease reaction described in step (2) are in condition: enzyme dosage: every 20 μ g glycoprotein makes
With the enzyme of 1U；Reaction temperature: 37 DEG C；Reaction time: 2h.
Pre-treating method above-mentioned, the chromatograph packing material that step (3) uses are that the Venusil HILIC of Beaune Ai Jieer is filled out
Material.
Pre-treating method above-mentioned will be lyophilized N- sugar chain after step (2), enter step after being redissolved using equilibrium liquid
(3)；The equilibrium liquid is the mixed solution of 80% (v) acetonitrile and 0.2% (v) trifluoroacetic acid.
Pre-treating method above-mentioned, which is characterized in that step (3) are as follows: after loading, wash impurity using equilibrium liquid, then use
0.1% (v) solution of trichloroacetic acid elutes N- sugar chain.
Pre-treating method above-mentioned, step (3) carry out in liquid-transfering gun pipette tips；Front end inside the liquid-transfering gun pipette tips is solid
Surely there is C8 Solid Phase Extraction disc；The disc plays sieve plate.
Pre-treating method above-mentioned removes the method for sialic acid described in step (4) are as follows: heat in trifluoroacetic acid environment
To 70-100 DEG C, 20-40min is maintained.
Further, the concentration of trifluoroacetic acid is 0.2% (v) in the trifluoroacetic acid environment.
The present invention also provides a kind of low costs, simple and quick glycoprotein N- sugar chain quantitative analysis method, before it includes
The pre-treating method stated, further includes:
Matrix-assisted laser desorption ionization analyzes N- sugar chain.Quantitative analysis method above-mentioned, it is described
The parameter of mass spectral analysis are as follows:
Mode: cation reflective-mode；
Acceleration voltage: 20kV；
Laser energy: 4500.
The invention has the following beneficial effects: firstly, the present invention N- sugar chain analysis before carried out asialoglycoprotein the step of,
The mass signal for enhancing N- sugar chain, increases the parsing abundance of sugar chain type.Further, the present invention is carried out using trifluoroacetic acid
Asialoglycoprotein processing, handles compared to sialidase, has saved time and economic cost.
Secondly, the present invention carries out purification enrichment to N- sugar chain using hydrophilic chromatographic filler, can be carried out in liquid-transfering gun pipette tips,
It is easy to operate.
Again, disclosed by the invention is widely suitable to the analyses of the N- sugar chain of the glycoprotein of separate sources, and analyze result and stablize
Reliably.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Detailed description of the invention
Fig. 1 is glycoprotein N- sugar chain analysis flow chart diagram of the invention.
Fig. 2 is the MALDI spectrogram of IgA1 obtained through present invention analysis.
Fig. 3 is the MALDI spectrogram of UMOD obtained through present invention analysis.
Fig. 4 is the MALDI spectrogram that the N- sugar chain of IgA1 is handled without asialoglycoprotein.
Fig. 5 is the MALDI spectrogram that the N- sugar chain of IgA1 is obtained through asialoglycoprotein enzymatic treatment post analysis.
Fig. 6 is the N- sugar chain quantitative repeatability assessment figure of IgA1.
Specific embodiment
It is as shown in Figure 1 experiment flow of the invention.As seen from the figure, glycoprotein needs to beat by denaturation, reductive alkylation
It disulfide bond is opened, removes N- sugar chain using PNGaseF proteolytic cleavage, super filter tube is collected by centrifugation N- sugar chain, and HILIC, which is enriched with, to be purified
N- sugar chain, acid treatment remove the sialic acid of N- sugar chain, Matrix-assisted laser desorption ionization (MALDI-TOF
MS) the N- sugar chain of asialoglycoprotein is analyzed.
Technical solution in order to enable those skilled in the art to better understand the present invention, with reference to the accompanying drawing to the present invention do into
One step detailed description.
The N- sugar chain analysis of glycoprotein Immunoglobulin alpha 1 (IgA1) in 1 human blood of embodiment
1. method
(1) IgA1 of the 100 μ g isolated and purified from human blood is dissolved in the ammonium hydrogen carbonate of the 50mmol/L of 200 μ L
(NH4HCO3) in solution, and be denaturalized in 100 DEG C of heating 10min.
(2) dithiothreitol (DTT) (DTT) of final concentration 20mmol/L is added in 56 DEG C of oscillating reactions 45min, final concentration is added
50mmol/L iodoacetamide (IAM) is protected from light 1h in room temperature.
(3) reaction solution is all added in 30KD super filter tube, and 13000g is centrifuged 15min × 3 time, every time with 200 μ L's
The NH of 50mmol/L4HCO3It rinses.
(4) NH of the 50mmol/L of 100 μ L is added4HCO3With the PNGase F protein enzyme solutions of 5U (1: 20), shake at 37 DEG C
The 2h that swings in device that the reaction was continued.
(5) 13000g is centrifuged 15min × 3 time, collects N- sugar chain, every time with the ultrapure water of 200 μ L.Collection liquid freeze-drying
It is spare afterwards.
(6) the HILIC filler of 5mg is weighed with EP pipe, first rinses filler 10min × 3 time with 0.1% trifluoroacetic acid (TFA),
80%ACN, 0.2%TFA solution equilibria 5min × 3 time are used again.
(7) the N- sugar chain being lyophilized in step (5) is used into 80%ACN, 0.2%TFA solution redissolves, and with step (6)
Filler mixing combines 2h in room temperature rotation.
(8) it is then transferred to 100 microlitres of liquid transfer gun heads (C8 Solid Phase Extraction disc is as sieve plate), 3000g is centrifuged 1min.
(9) with 80% acetonitrile (ACN), 0.2%TFA solution washes the impurity of primary removal non-specific binding again, finally uses
0.1%TFA is eluted 3 times, the N- sugar chain purified.It is saved backup for -20 DEG C after freeze-drying.
(10) the N- sugar chain for the purifying for obtaining step (9) enrichment, is added the 0.2% trifluoroacetic acid solution weight of 200 μ L
New dissolution, 80 DEG C of oscillation heating 30min remove sialic acid.What finally freeze-drying obtained is the N- sugar chain for removing sialic acid.
(11) a pipe peptide fragment calibration standard product is dissolved with the TA30 of 125 μ L (acetonitrile: 0.1%TFA=30: 70 (v/v))
(Peptide Calibration Standard II) is used for instrumental correction.2, the 5- dihydroxy benzenes of 20mg/ml is prepared with TA30
Formic acid (2,5-DHB).The ultrapure water of 10 μ L carries out the N- sugar chain of dissolving step (10).
(12) it takes 1 μ L sample point on Anchor Chip target, after natural drying, takes 1 μ L matrix point on sample target, from
So after drying, it is pushed into be measured in ion source.Outer calibration is carried out to instrument using Peptide Calibration Standard II
Just, relative error≤10ppm is calibrated to.
(13) N sugar chain sample is analyzed, using MALDI-TOF mass spectrometer (Bruker Daltonics company), cation
Reflective-mode, acceleration voltage 20kV, laser energy 4500, each sample parallel acquisition three open spectrogram.
(14) initial data is analyzed using 3.3 version software of flexAnalysis, calculates each sugar chain peak intensity, as
The content foundation of the sugar-type.When calculating sugar-type ratio, the relative amount of each sugar-type is first calculated after normalization again.
2. result
The MALDI-TOF spectrogram that the N- sugar chain of three groups of human plasma IgA1 obtains is as shown in Figure 2, the results showed that, energy of the present invention
Realize effectively N- sugar chain quantitative analysis.
The N- sugar chain analysis of glycoprotein urine heregulin (UMOD) in 2 human urine of embodiment
1. method
(1) UMOD for the 100 μ g that human urine isolates and purifies is dissolved in the ammonium hydrogen carbonate of the 50mmol/L of 200 μ L
(NH4HCO3) it in solution, and is denaturalized in 100 DEG C of heating 10min.
(2) dithiothreitol (DTT) (DTT) of final concentration 20mmol/L is added in 56 DEG C of oscillating reactions 45min, final concentration is added
50mmol/L iodoacetamide (IAM) is protected from light 1h in room temperature.
(3) reaction solution is all added in 30KD super filter tube, and 13000g is centrifuged 15min × 3 time, every time with 200 μ L's
The NH4HCO3 of 50mmol/L is rinsed.
(4) the PNGaseF protein enzyme solution of the NH4HCO3 and 5U (1: 20) of the 50mmol/L of 100 μ L is added, shakes at 37 DEG C
The 2h that swings in device that the reaction was continued.
(5) 13000g is centrifuged 15min × 3 time, collects N- sugar chain, every time with the ultrapure water of 200 μ L.Collection liquid freeze-drying
It is spare afterwards.
(6)~Venusil HILIC the filler (Beaune Ai Jieer) of 5mg is weighed with EP pipe, is first filled out with 0.1%TFA flushing
Expect 10min × 3 time, then with 80%ACN, 0.2%TFA solution equilibria 5min × 3 time.
(7) the N- sugar chain being lyophilized in step (5) is used into 80%ACN, 0.2%TFA solution redissolves, and with step (6)
Filler mixing combines 2h in room temperature rotation.
(8) it is then transferred to 100 microlitres of liquid transfer gun heads (C8 Solid Phase Extraction disc is as sieve plate), 3000g is centrifuged 1min.
(9) 80%ACN is used, 0.2%TFA solution washes the impurity of primary removal non-specific binding again, finally uses 0.1%TFA
Elution 3 times, the N- sugar chain purified.It is saved backup for -20 DEG C after freeze-drying.
(10) the N- sugar chain for the purifying for obtaining step (9) enrichment, is added the 0.2% trifluoroacetic acid solution weight of 200 μ L
New dissolution, 80 DEG C of oscillation heating 30min remove sialic acid.What finally freeze-drying obtained is the N- sugar chain for removing sialic acid.
(11) a pipe Peptide Calibration Standard II is dissolved with the TA30 of 125 μ L be used for instrumental correction.
The 2,5-DHB of 20mg/ml are prepared with TA30.The ultrapure water of 10 μ L carries out the N- sugar chain of dissolving step (10).
(12) it takes 1 μ L sample point on Anchor Chip target, after natural drying, takes 1 μ L matrix point on sample target, from
So after drying, it is pushed into be measured in ion source.Outer calibration is carried out to instrument using Peptide Calibration Standard II
Just, relative error≤10ppm is calibrated to.
(13) N sugar chain sample is analyzed, using MALDI-TOF mass spectrometer (Bruker Daltonics company), cation
Reflective-mode, acceleration voltage 20kV, laser energy 4500, each sample parallel acquisition three open spectrogram.
(14) initial data is analyzed using 3.3 version software of flexAnalysis, calculates each sugar chain peak intensity, as
The content foundation of the sugar-type.When calculating sugar-type ratio, the relative amount of each sugar-type is first calculated after normalization again.
2. result
As shown in figure 3, the present invention still can effective quantitative analysis N- sugar chain from urine UMOD.Show method of the invention
Wide adaptability.
The beneficial effect generated in the form of comparative example to processing mode of the present invention to sialic acid is done further below
Explanation.
Comparative example 1 does not do the N- sugar chain quantitative analysis of sialic acid removal
1. method
With the method for embodiment 1, but to skip step (10).
2. result
Mass spectrogram can make the mass signal of N- sugar chain weak as shown in figure 4, not removing sialic acid, the sugar chain type of identification
It is few.
Comparative example 2 removes the N- sugar chain quantitative analysis of sialic acid using sialidase
1. method
" sialidase processing " is replaced with the method for embodiment 1, but by step (10).
2. result
As shown in figure 5, asialoglycoprotein enzyme specific can remove sialic acid, the signal of sugar chain can be dramatically increased, be identified
Sugar chain type also will increase.But asialoglycoprotein enzyme is at high cost, the holding time is short, and the reaction time needs overnight.
Further, the present invention has preferable stability, is illustrated below with the form of experimental example to this.
Experimental example stability test
1. method
The blood plasma for taking 3 parts of different peoples, is tested according to the method for embodiment 1.
2. result
As shown in fig. 6, the N- sugar chain Species differences very little for the IgA1 that three groups of different peoples obtain, shows that this method is very stable,
Favorable reproducibility, high reliablity.
To sum up, method of the invention can simply, quickly and efficiently realize the quantitative analysis of glycoprotein N- sugar chain, and divide
It analyses that at low cost, the scope of application is wider, there is good industrialization prospect.
Claims (13)
1. the pre-treating method of a kind of low cost, simple and quick glycoprotein N- sugar chain quantitative analysis, which is characterized in that it includes
Following steps:
(1) reductive alkylation processing is carried out after glycoprotein heat denatured；
(2) the N- sugar chain on glycopeptide is removed using the digestion of PNGase F protein in super filter tube, N- sugar chain is collected by centrifugation；
(3) hydrophilic Interaction Chromatography purification enrichment N- sugar chain is used；
(4) after removing sialic acid, dry concentration.
2. pre-treating method as described in claim 1, which is characterized in that the processing of reductive alkylation described in step (1) are as follows: point
It Shi Yong not dithiothreitol (DTT) and iodoacetamide processing.
3. method according to claim 2, which is characterized in that the dithiothreitol (DTT) processing are as follows: 50-60 DEG C of oscillating reactions
30-60min。
4. pre-treating method as claimed in claim 2, which is characterized in that the iodoacetamide processing are as follows: final concentration 30- is added
70mmol/L iodoacetamide is protected from light 0.5-1.5h in room temperature.
5. pre-treating method as described in claim 1, which is characterized in that endonuclease reaction described in step (2) is in condition: enzyme
Dosage: every 20 μ g glycoprotein uses the enzyme of 1U；Reaction temperature: 37 DEG C；Reaction time: 2h.
6. pre-treating method as described in claim 1, which is characterized in that the chromatograph packing material that step (3) uses is that Beaune love is outstanding
Your Venusil HILIC filler.
7. pre-treating method as described in claim 1, which is characterized in that be lyophilized, use to N- sugar chain after step (2)
Equilibrium liquid enters step (3) after redissolving；The equilibrium liquid is the mixed solution of 80% (v) acetonitrile and 0.2% (v) trifluoroacetic acid.
8. pre-treating method as claimed in claim 7, which is characterized in that step (3) are as follows: after loading, washed using equilibrium liquid miscellaneous
Matter, then N- sugar chain is eluted with 0.1% (v) solution of trichloroacetic acid.
9. pre-treating method a method as claimed in any one of claims 1-8, which is characterized in that step (3) carries out in liquid-transfering gun pipette tips；
C8 Solid Phase Extraction disc is fixedly arranged at the front end with inside the liquid-transfering gun pipette tips；The disc plays sieve plate.
10. pre-treating method as described in claim 1, which is characterized in that remove the method for sialic acid described in step (4)
Are as follows: it is heated to 70-100 DEG C in trifluoroacetic acid environment, maintains 20-40min.
11. pre-treating method as claimed in claim 7, which is characterized in that trifluoroacetic acid is dense in the trifluoroacetic acid environment
Degree is 0.2% (v).
12. a kind of low cost, simple and quick glycoprotein N- sugar chain quantitative analysis method, which is characterized in that it includes such as right
It is required that any pre-treating method of 1-11, further includes:
Matrix-assisted laser desorption ionization analyzes N- sugar chain.
13. quantitative analysis method as claimed in claim 12, which is characterized in that the parameter of the mass spectral analysis are as follows:
Mode: cation reflective-mode；
Acceleration voltage: 20kV；
Laser energy: 4500.
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