CN105833848A - Filler for separating sulfydryl and non-sulfydryl peptide fragments and preparation method thereof - Google Patents
Filler for separating sulfydryl and non-sulfydryl peptide fragments and preparation method thereof Download PDFInfo
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- CN105833848A CN105833848A CN201610163374.4A CN201610163374A CN105833848A CN 105833848 A CN105833848 A CN 105833848A CN 201610163374 A CN201610163374 A CN 201610163374A CN 105833848 A CN105833848 A CN 105833848A
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- Prior art keywords
- sulfydryl
- peptide fragment
- filler
- separating
- preparation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
-
- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the technical field of separation and analysis, and in particular, relates to a filler for separating a sulfydryl-containing peptide fragment and a sulfydryl-free peptide fragment and a preparation method thereof. The method comprises the basic steps: firstly, mixing trifluoroethylene sulfonic acid group-containing polymer material and cysteine, placing the mixture in an alkaline buffer system, and incubating; centrifuging to obtain a cysteine-modified polymer material, washing with water for a plurality of times, then incubating the material with a pre-synthesized nano gold colloid, and centrifuging to obtain a nano gold-modified polymer material; then filling a capillary tube or other columns with the material, and thus obtaining a column for separating the sulfydryl-containing peptide fragment and the sulfydryl-free peptide fragment. The separation filler prepared by using the method is novel in method, simple in preparation process, and easy to restore and repeatedly use, can be applied to removal of the sulfydryl-containing peptide fragment, and is used for separation and analysis of the sulfydryl-free peptide fragment in effluent; the filler can also be used for capture and elution of the sulfydryl-containing peptide fragment, and is used for separation and analysis of the sulfydryl-containing peptide fragment in eluate.
Description
Technical field
The invention belongs to separate analytical technique field, be specifically related to a kind of for separating containing sulfydryl peptide fragment and the filler without sulfydryl peptide fragment and preparation method thereof.
Background technology
In proteomics research, in the identification and analysis of the peptide fragment that mass-spectrometric technique is formed after being widely used in proteolysis, mainly include LC-MS technology, substance assistant laser desorpted ionization time of flight etc..And when sample complexity, need the sample pretreatment before sample is carried out mass spectrum, wherein, the pre-separation enrichment to target analytes is very important a kind of sample preprocessing means.
Meanwhile, in protein N-terminal analysis method, need the separation method developing efficient end with non-end peptide fragment, to realize the efficient qualification of terminal peptide fragment.Wherein, a most crucial step is, the non-end peptide fragment after utilizing material to derive sulfydryl separates.There is important scientific meaning containing sulfydryl peptide fragment because of it it addition, endogenic, also have relevant document to report, based on sulfydryl exchange or other deriving methods, it is carried out separation and concentration, and correlation technique step is complicated and the longest.Therefore, develop efficient chromatographic isolation filler and sulfydryl and non-sulfydryl peptide fragment are carried out separation and concentration, it is possible to advantageously promote the research of proteomics.
Summary of the invention
It is an object of the invention to provide a kind of preparation process simply, easily to bring back to life and reusable for separating containing sulfydryl peptide fragment and the filler not containing sulfydryl peptide fragment and preparation method thereof.
The preparation method of the filler separating sulfydryl and non-sulfydryl peptide fragment that the present invention provides, it concretely comprises the following steps: first, will mix with cysteine containing trifluoro ethyl sulfonic acid based high-polymer material, is placed in the buffer system of alkalescence and hatches；Then, by the centrifugal polymeric material obtaining cysteine modified, wash through several times；Again this material is hatched with pre-synthesis nano Au colloid, centrifugal, obtain the high polymer material of decorated by nano-gold.
This material is inserted in the pillars such as capillary tube, i.e. obtains for containing sulfydryl and the pillar without sulfydryl peptides separation.
Further, the concrete operations flow process of the present invention is as follows:
(1) cysteine of 1 unit mass is dissolved in the buffer system without ammonia of alkalescence；
(2) the trifluoro ethyl sulfonic acid sill weighing 0.1-5 unit mass mixes with above-mentioned solution, is placed at a temperature of 25 DEG C-60 DEG C, reacts 1-24 hour；
(3) after reaction terminates, by centrifugal segregation residual reaction liquid, then with deionized water cleaning material more than 2 times, stand-by；
(4) cleaned material is mixed with synthetic gold size solution, concussion reaction under arbitrary temp (more than 0 DEG C, less than 90 DEG C), until gold goal absorption is saturated；
(5), after using deionized water cleaning material several times, material is dried, i.e. obtains for separating sulfydryl and the filler of non-sulfydryl peptide fragment.
In the present invention, a diameter of 60-70 micron of described preferred filler.
The present invention, according to different use scenes, can be used directly for during in biased sample, sulfydryl separates with non-sulfydryl peptide fragment, it is also possible to according to using needs, is inserted in capillary column or conventional post, carries out on-line chromatograph separation.
Filler in the present invention can use the method for eluting to bring back to life, and is used multiple times.
Utilize the inventive method can promote the research of proteomics with the sulfydryl of synthesizing efficient and non-sulfydryl peptide fragment separating filler.
Accompanying drawing explanation
Fig. 1 is that sulfydryl is schemed with non-sulfydryl peptide fragment mixing MALDI-ToF MS.
Fig. 2 is that the MALDI-ToF MS of effluent schemes (without the peptide fragment of sulfydryl) after filler separates.
Fig. 3 is that the MALDI-ToF MS of the eluent after filler eluting schemes (peptide fragment containing sulfydryl).
Fig. 4 is the field emission scanning electron microscope figure of (b) after filler is modified (a) before gold goal and modified gold goal.
Fig. 5 is two batches of fillers (right side one, the two) comparison diagram after filler is modified (the first from left) before gold goal, nano gold spherical (the second from left) and modified gold goal.
Fig. 6 is preparation flow of the present invention diagram.
Detailed description of the invention
Embodiment 1:
What the present invention provided separates sulfydryl and the filler of non-sulfydryl peptide fragment, can separate sulfydryl and non-sulfydryl peptide fragment, and the peptide fragment after separating can be analyzed by binding matrix assisted laser desorption flight time mass spectrum:
(1) being inserted by this filler in the capillary column of 320 micron inside diameter, pack length is 10 centimetres；
(2) by 50 μ L sulfydryl peptide fragments and non-sulfydryl peptide fragment mixed liquor with 1
The speed of μ L/min passes through capillary column, collects effluent, obtains non-sulfydryl peptide fragment；
(3) it is passed through 100 μ L water with the speed of 2 μ L/min and rinses pillar；
(4) it is passed through 80% acetonitrile solution (containing 0.1% trifluoroacetic acid) with 2 μ L/min speed, sulfydryl peptide fragment is carried out eluting, collect eluent, obtain containing sulfydryl peptide fragment；
(5) by effluent and eluent point sample, enter substance assistant laser desorpted flight time mass spectrum and identify.
Embodiment 2:
What the present invention provided separates sulfydryl and the filler of non-sulfydryl peptide fragment, can separate sulfydryl and non-sulfydryl peptide fragment, and the peptide fragment after separating can be analyzed by binding matrix assisted laser desorption flight time mass spectrum:
(1) being inserted by this filler in the capillary column of 250 micron inside diameter, pack length is 5 centimetres；
(2) by 10 μ L sulfydryl peptide fragments and non-sulfydryl peptide fragment mixed liquor with 0.15
The speed of μ L/min passes through capillary column, collects effluent, obtains non-sulfydryl peptide fragment；
(3) it is passed through 20 μ L water with the speed of 2 μ L/min and rinses pillar；
(4) it is passed through 80% acetonitrile solution (containing 0.1% trifluoroacetic acid) with 2 μ L/min speed, sulfydryl peptide fragment is carried out eluting, collect eluent, obtain containing sulfydryl peptide fragment；
(5) by effluent and eluent point sample, enter substance assistant laser desorpted flight time mass spectrum and identify.
Claims (4)
1. the preparation method of the filler separating sulfydryl and non-sulfydryl peptide fragment, it is characterised in that concretely comprise the following steps: first, will mix with cysteine containing trifluoro ethyl sulfonic acid based high-polymer material, be placed in the buffer system of alkalescence and hatch；Then, by the centrifugal polymeric material obtaining cysteine modified, wash through several times；Again this material is hatched with pre-synthesis nano Au colloid, centrifugal, obtain the high polymer material of decorated by nano-gold.
The preparation method of the separating filler of sulfydryl the most according to claim 1 and non-sulfydryl peptide fragment, it is characterised in that concrete operations flow process is as follows:
(1) cysteine of 1 unit mass is dissolved in the buffer system without ammonia of alkalescence；
(2) the trifluoro ethyl sulfonic acid sill weighing 0.1-5 unit mass mixes with above-mentioned solution, is placed at a temperature of 25 DEG C-60 DEG C, reacts 1-24 hour；
(3) after reaction terminates, by centrifugal segregation residual reaction liquid, then with deionized water cleaning material more than 2 times, stand-by；
(4) cleaned material is mixed with synthetic gold size solution, concussion reaction at a temperature of 0 DEG C-90 DEG C, until gold goal absorption is saturated；
(5), after using deionized water cleaning material several times, material is dried, i.e. obtains for separating sulfydryl and the filler of non-sulfydryl peptide fragment.
3. by preparation method described in claim 1 or 2 prepare for separating sulfydryl and the filler of non-sulfydryl peptide fragment.
4. filler as claimed in claim 3 sulfydryl and application in non-sulfydryl peptides separation in biased sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610163374.4A CN105833848A (en) | 2016-03-19 | 2016-03-19 | Filler for separating sulfydryl and non-sulfydryl peptide fragments and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610163374.4A CN105833848A (en) | 2016-03-19 | 2016-03-19 | Filler for separating sulfydryl and non-sulfydryl peptide fragments and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105833848A true CN105833848A (en) | 2016-08-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610163374.4A Pending CN105833848A (en) | 2016-03-19 | 2016-03-19 | Filler for separating sulfydryl and non-sulfydryl peptide fragments and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117825481A (en) * | 2023-12-29 | 2024-04-05 | 安徽省疾病预防控制中心(省健康教育所 省公共卫生研究院) | Derived mass spectrometry imaging method for free cysteine (Cys) in tissue section |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083515A3 (en) * | 2000-04-28 | 2002-07-04 | Accurate Polymers Inc | Simulated activity of protein a displayed by ligands attached to a cellulose bead surface |
| CN103234794A (en) * | 2013-04-20 | 2013-08-07 | 复旦大学 | Zip-Tips for trapping sulfhydryl-containing peptide segment and preparation method thereof |
| CN105319116A (en) * | 2015-11-18 | 2016-02-10 | 复旦大学 | Protein N-terminal peptide fragment separation method based on magnetic microsphere |
-
2016
- 2016-03-19 CN CN201610163374.4A patent/CN105833848A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083515A3 (en) * | 2000-04-28 | 2002-07-04 | Accurate Polymers Inc | Simulated activity of protein a displayed by ligands attached to a cellulose bead surface |
| CN103234794A (en) * | 2013-04-20 | 2013-08-07 | 复旦大学 | Zip-Tips for trapping sulfhydryl-containing peptide segment and preparation method thereof |
| CN105319116A (en) * | 2015-11-18 | 2016-02-10 | 复旦大学 | Protein N-terminal peptide fragment separation method based on magnetic microsphere |
Non-Patent Citations (1)
| Title |
|---|
| LANTING LI ET AL.: "Isolation of acetylated and free N-terminal peptides from proteomic samples based on tresyl-functionalized microspheres", 《TALANTA》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117825481A (en) * | 2023-12-29 | 2024-04-05 | 安徽省疾病预防控制中心(省健康教育所 省公共卫生研究院) | Derived mass spectrometry imaging method for free cysteine (Cys) in tissue section |
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Application publication date: 20160810 |
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