CN116626189B - Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS - Google Patents
Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS Download PDFInfo
- Publication number
- CN116626189B CN116626189B CN202310559905.1A CN202310559905A CN116626189B CN 116626189 B CN116626189 B CN 116626189B CN 202310559905 A CN202310559905 A CN 202310559905A CN 116626189 B CN116626189 B CN 116626189B
- Authority
- CN
- China
- Prior art keywords
- reference sample
- standard
- dna
- solution
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 76
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 title claims abstract description 59
- 230000005778 DNA damage Effects 0.000 title claims abstract description 41
- 231100000277 DNA damage Toxicity 0.000 title claims abstract description 41
- 238000004458 analytical method Methods 0.000 title claims abstract description 30
- 239000013074 reference sample Substances 0.000 claims abstract description 154
- 239000000126 substance Substances 0.000 claims abstract description 61
- 238000001514 detection method Methods 0.000 claims abstract description 56
- 238000011084 recovery Methods 0.000 claims abstract description 50
- 239000000523 sample Substances 0.000 claims abstract description 33
- 239000012086 standard solution Substances 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000013642 negative control Substances 0.000 claims description 14
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 12
- 238000000132 electrospray ionisation Methods 0.000 claims description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- -1 p-toluenesulfonyl group Chemical group 0.000 claims description 7
- 125000006239 protecting group Chemical group 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 230000003247 decreasing effect Effects 0.000 claims description 5
- 238000000611 regression analysis Methods 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 claims description 3
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims 3
- 238000006757 chemical reactions by type Methods 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 40
- 239000008367 deionised water Substances 0.000 description 28
- 229910021641 deionized water Inorganic materials 0.000 description 28
- 239000000203 mixture Substances 0.000 description 17
- 238000006482 condensation reaction Methods 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 238000011529 RT qPCR Methods 0.000 description 12
- 238000004364 calculation method Methods 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 238000001556 precipitation Methods 0.000 description 9
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000007480 sanger sequencing Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- MZYHCMVDZHPUST-UHFFFAOYSA-N 2-[cyclopropyl(9h-fluoren-9-ylmethoxycarbonyl)amino]acetic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N(CC(=O)O)C1CC1 MZYHCMVDZHPUST-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000007086 side reaction Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000005945 translocation Effects 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 2
- 239000012346 acetyl chloride Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 150000001793 charged compounds Chemical class 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000002716 delivery method Methods 0.000 description 2
- 230000027832 depurination Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 241001156002 Anthonomus pomorum Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- WPUTYTINVUOURJ-UHFFFAOYSA-N N-methyl-2-[2-[2-(2-propoxyethoxy)ethoxy]ethoxy]ethanamine Chemical compound CCCOCCOCCOCCOCCNC WPUTYTINVUOURJ-UHFFFAOYSA-N 0.000 description 1
- 108091060592 XDNA Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UYCAUPASBSROMS-AWQJXPNKSA-M sodium;2,2,2-trifluoroacetate Chemical compound [Na+].[O-][13C](=O)[13C](F)(F)F UYCAUPASBSROMS-AWQJXPNKSA-M 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- 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)
Abstract
The application relates to the field of DNA coding compound libraries, and particularly discloses an analysis method for DNA damage in an On-DNA chemical reaction based On LC-MS. The analysis method comprises the following steps: selecting an internal reference sample and a standard substance, and preparing a group of standard liquids containing the standard substance and the internal reference sample; carrying out LC-MS analysis on the standard solution, and obtaining a standard working curve by taking the molar ratio of the standard substance to the internal reference sample as an abscissa and the response ratio of the detection signal as an ordinate; and (3) carrying out On-DNA chemical reaction On the reference sample to obtain a sample liquid to be detected, mixing the sample liquid with a standard substance, carrying out LC-MS analysis, calculating the recovery rate of the reference sample, and judging the DNA damage condition. The analysis method can finish DNA damage identification of hundreds of reaction types in one day through the change of the molecular weight and the signal intensity of a sample provided by LC-MS, which is of great significance to the later screening and the high-throughput analysis of DEL libraries.
Description
Technical Field
The application relates to the technical field of DNA coding compound libraries, in particular to an analysis method for DNA damage in On-DNA chemical reaction based On LC-MS.
Background
The DNA coding library (DELs) was originally a concept proposed by Brenner and Lerner in 1992 and has become an important recognition tool in early drug screening discovery. DELs have unique DNA tags per molecule, and millions to billions of compounds can be screened rapidly and in parallel. In recent years, synthetic methods for constructing DEL libraries (also known as On-DNA chemistry) have evolved rapidly, and extensive reactions from organic chemistry have now been developed. However, with the increase in the types of reactions, side reactions with DNA are also continuously occurring. As part of the diversity of DNA sequence coding, modified bases can cause various DNA damage including mutation, translocation, fragmentation, etc., ultimately resulting in significant loss of amplified coding DNA, resulting in termination of the post PCR process, thereby reducing apparent DNA concentration and negatively affecting signal.
Several methods have been developed to quantify the partial DNA damage caused by chemical reactions, most of the traditional methods of detecting DNA damage are either global, sequence independent, or limited to one or several defined types of DNA damage. Such as Sanger sequencing and qPCR, which are currently well known.
Sanger sequencing refers to the addition of dideoxynucleotide triphosphate (ddNTP) during DNA replication to produce a series of end-terminated DNA strands, the DNA length and sequence of which can be read by electrophoresis and corresponding instrumentation. Sanger sequencing can report significant point mutations, but it is not able to quantify less frequent point mutations and is therefore not applicable to DEL libraries of only tens of bp in DNA sequence.
QPCR is a method of adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in real time by utilizing fluorescent signal accumulation, and finally quantitatively analyzing an unknown template through a standard curve. However, this method has a great disadvantage, such as: qpcr requires normalization of the concentrations of standard and sample at the initial stage. However, the sample is damaged after chemical reaction, and the molecular weight may be changed, so that the sample is often a mixture, and the concentration of the sample cannot be accurately measured. The amplification efficiency and specificity of qPCR are related to many factors (primers, amplification temperature, amplicon length, etc.), so qPCR in practice often requires a lot of manpower and resources to optimize the procedure, which is unsuitable for several hundred DNA chemical reactions that require a lot of labor to determine the amplification efficiency and standardization procedure of a single sample, and is unsuitable for high throughput analysis. qPCR provides only one relative recovery rate and cannot analyze specific DNA damage types, such as single or multiple base mutations in the amplicon, which do not affect qPCR amplification.
In view of this, there is a strong need for an analytical method that can rapidly and accurately quantify the partial DNA damage caused by chemical reactions.
Disclosure of Invention
In order to solve the technical problems, the application provides an analysis method for DNA damage in On-DNA chemical reaction based On LC-MS, which is used for detecting and analyzing by LC-MS, can rapidly calculate the recovery rate of On-DNA compounds by an external standard method, and simultaneously can finish DNA damage identification of hundreds of reaction types in one day due to the change of the molecular weight and the signal intensity of a sample provided by LC-MS as an internal reference sample and a standard substance do not participate in any chemical reaction, which is significant for later screening and high-throughput analysis of DEL library.
The application adopts the following technical scheme:
In a first aspect, the present application provides a method for analysis of DNA damage in an On-DNA chemical reaction based On LC-MS, comprising:
Selecting a DNA label for carrying out On-DNA chemical reaction as an internal reference sample;
selecting a specific DNA fragment as a standard, wherein the LC-MS spectrum of the standard is not coincident with the detection signal response in the LC-MS spectrum of the reference sample;
Preparing a group of standard solutions containing standard substances and internal reference samples, wherein the molar ratio of the standard substances to the internal reference samples in the standard solutions is sequentially decreased; carrying out LC-MS analysis on the standard solution, carrying out regression analysis by taking the molar ratio of the standard substance to the internal reference sample as an abscissa and the ratio of the detection signal response of the LC-MS of the standard substance to the internal reference sample as an ordinate, so as to obtain a standard working curve;
And (3) carrying out On-DNA chemical reaction On the reference sample, carrying out pretreatment On the reaction liquid to obtain a sample liquid to be detected, mixing the sample liquid with a standard substance, carrying out LC-MS analysis under the same condition to obtain a detection signal response, carrying the detection signal response into a standard working curve, calculating the quantity of substances of the reference sample in the sample liquid to be detected, thus obtaining the recovery rate of the reference sample, and judging the DNA damage condition of the reference sample through the recovery rate and the change of the detection signal response of the reference sample.
Further, in the preparation of the standard working curve, the detection signal response is the peak area or peak height of the UV signal peak and the Mass signal peak in the LC-MS detection spectrum.
Further, in the production of the standard working curve, the ratio of the detection signal response on the ordinate=the (UV peak area×mass peak area) of the standard/the (UV peak area×mass peak area) of the reference sample.
Further, both the standard and the reference sample are DNA or On-DNA compounds, and the separation degree of the liquid chromatographic peak of the reference sample and the standard is >1.5, and the mass spectrum peak is not coincident.
Further, in the process of preparing the reference sample, amino protecting group is used for amino protecting treatment of the DNA tag.
Further, the amino protecting group used in preparing the reference sample includes at least one of acetyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, and nitrobenzenesulfonyl.
Further, the structure of the internal reference sample is:
Further, the amount of the substance of the standard in the standard solution is 1pmol to 1nmol, and the amount of the substance of the internal reference sample is 1pmol to 100nmol.
Further, the analysis method further includes:
Preparing a negative control solution containing an internal reference sample, mixing the negative control solution with a standard substance, performing LC-MS analysis under the same condition to obtain detection signal intensity, taking the detection signal intensity into a standard working curve, and calculating to obtain a negative recovery rate.
Further, the chromatographic conditions of LC-MS are:
Mobile phase a: is obtained by dissolving hexafluoroisopropanol, diisopropylethylamine and EDTA in water;
mobile phase B: methanol;
Flow rate: 0.3-0.8 mL/min;
Detection wavelength: 250-270 nm;
The temperature of the electrospray ionization probe is 280-320 ℃, and the source temperature is 330-370 ℃;
The ESI capillary is-18 to-22 kV;
The mass detector operates in a negative ion full scan mode in the 700-2000 (m/z) range.
In summary, the application has the following beneficial effects:
1. According to the analysis method provided by the application, a standard working curve capable of representing the linear relation between a standard substance and an internal reference sample is prepared by an LC-MS and an external standard method, and the recovery rate of the internal reference sample after the On-DNA chemical reaction is carried out by using the internal reference sample is quantitatively analyzed.
2. Since the DNA tag in the reference sample of the present application does not participate in chemical reaction, the change in molecular weight of the reference sample is derived from a change (mutation, translocation, cleavage) of DNA strand, not a change in chemical structure of DNA. Therefore, various damage conditions generated On the DNA of the reference sample in the On-DNA chemical reaction can be qualitatively analyzed through the change of the detection signal response of the reference sample (such as the charge-to-Mass ratio of molecular ion peaks in Mass, the intensity of UV peaks and the like).
3. As a result of On-DNA chemistry, side reactions with DNA often occur, resulting in changes in DNA. This means that when LC-MS is used to analyze the recovery rate of DNA in a sample to be measured, it is impossible to select a standard substance having exactly the same structure as that of the sample to be measured for calibration as in the conventional external standard method. Therefore, the standard solution is prepared by simultaneously selecting the standard substance and the internal reference sample, the molar ratio of the standard substance to the internal reference sample is taken as an abscissa, and the ratio of the LC-MS detection signal response of the standard substance to the internal reference sample is taken as an ordinate, so that a standard working curve is obtained, and the correlation is good.
4. Compared with the existing Sanger sequencing and qPCR analysis method, the method is simple to operate, strong in specificity and short in time consumption, can analyze tens to hundreds of reaction types at the same time, and provides a quick and convenient effective way for DEL library synthesis efficiency and quality identification.
Drawings
FIG. 1 is an HPLC chart of Standard 1 in example 1;
FIG. 2 is an HPLC chart of reference sample S1 in example 1;
FIG. 3 is a standard operating curve 1 produced in example 1;
FIG. 4 is an HPLC chromatogram of the negative control in example 1;
FIG. 5 is an HPLC chart after the condensation reaction of DNA-NH 2 in example 1;
FIG. 6 is an HPLC chart after the condensation reaction of reference sample S1 in example 1;
FIG. 7 is an HPLC chart of reference sample S2 in example 2;
FIG. 8 is a standard operating curve 2 produced in example 2;
FIG. 9 is an HPLC chromatogram of the negative control in example 2;
FIG. 10 is an HPLC chart after the condensation reaction of reference sample S2 in example 2;
FIG. 11 is a Mass spectrum of the reference sample S2 in example 3 after the Boc removal reaction (sodium trifluoroacetate).
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The technical scheme of the invention is as follows:
a method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS, comprising the steps of:
(1) The DNA tag of the On-DNA chemical reaction was selected as an internal reference sample.
The internal sample may be any DNA or On-DNA compound, which contains a DNA tag for On-DNA chemical reaction to indicate whether side reaction with the DNA tag occurs during On-DNA chemical reaction, and various DNA damages such as mutation, translocation, cleavage, etc. of the DNA tag occur. By using the internal reference sample, tens to hundreds of reaction types which can occur on the DNA tag can be analyzed simultaneously, the application range is wide, and the identification of the synthesis efficiency and the quality on the DEL library can be carried out rapidly and conveniently.
Further, in the process of preparing the reference sample, amino protecting group is used for amino protecting treatment of the DNA tag. Namely, a DNA tag with an amino protecting group is selected as an internal reference sample.
Further, the amino protecting group used in preparing the reference sample includes at least one of acetyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, o (p) nitrobenzenesulfonyl.
Preferably, the structure of the internal reference sample S1 is as follows:
preferably, the structure of the internal reference sample S2 is as follows:
(2) And selecting a specific DNA fragment as a standard substance, preparing a group of standard solutions containing the standard substance and the internal reference sample, wherein the molar ratio of the standard substance to the internal reference sample in the standard solutions is sequentially decreased.
The standard substance can be any DNA or On-DNA compound, and the LC-MS spectrum of the standard substance is obviously different from the detection signal response position of the LC-MS spectrum of the reference sample, and the detection signal response position is specifically shown as follows: the LC chromatographic peak R (degree of separation) of both was >1.5, and the molecular weights of both were calculated by ProMass HR 2.0 to distinguish between the two, without functional groups capable of participating in chemical reactions.
Preferably, the structure of standard 1 is as follows:
the standard 1 is purchased from the company of biosciences, kirsry, and the product name is DNA initial fragment, and the specific sequence is:
5′-/5Phos/GAGTCA/iSp9/iUniAmM/iSp9/TGACTCCC-3′
further, the amount of the substance of the standard in the standard solution in which the standard and the reference sample S1 are mixed is 1pmol to 1nmol, and in a more preferred embodiment, the absolute amount of the substance of the standard is 80 pmol to 120pmol, and more preferably 100pmol.
Further, the amount of the substance of the reference sample S1 in the standard solution in which the standard substance and the reference sample S1 are mixed is 1pmol to 100nmol, and in a more preferred embodiment, the absolute amount of the substance of the reference sample S1 is 1pmol to 1nmol, and more preferably 50pmol, 100pmol, 200pmol, 300pmol, 400pmol, and 500pmol.
Further, the amount of the substance of the standard in the standard solution in which the standard and the reference sample S2 are mixed is 1pmol to 100nmol, and in a more preferred embodiment, the absolute amount of the substance of the standard is 1pmol to 1nmol, and more preferably, 50pmol, 100pmol, 200pmol, 300pmol, 400pmol, and 500pmol.
Further, the amount of the substance of the reference sample S2 in the standard solution in which the reference sample S2 and the reference sample S2 are mixed is 1pmol to 1nmol, and in a more preferred embodiment, the absolute amount of the substance of the reference sample S2 is 80 pmol to 120pmol, and more preferably 100pmol.
(3) And carrying out LC-MS analysis on the standard solution, carrying out regression analysis by taking the molar ratio of the standard substance to the internal reference sample as an abscissa and taking the ratio of the detection signal response of the LC-MS of the standard substance to the internal reference sample as an ordinate, and obtaining a standard working curve.
Further, in the preparation of the standard working curve, the detection signal response is the peak area or peak height of the UV signal peak and the Mass signal peak in the LC-MS detection spectrum.
More preferably, in the preparation of the standard working curve, the ratio of the detection signal response on the ordinate=the (UV peak area×mass peak area) of the standard/the (UV peak area×mass peak area) of the internal reference sample.
Further, the chromatographic conditions of LC-MS are:
Mobile phase a: is obtained by dissolving Hexafluoroisopropanol (HFIP), diisopropylethylamine (DIPEA) and EDTA in water; mobile phase B: methanol;
Flow rate: 0.3-0.8 mL/min;
Detection wavelength: 250-270 nm;
The temperature of the electrospray ionization probe is 280-320 ℃, and the source temperature is 330-370 ℃;
The ESI capillary is-18 to-22 kV;
The mass detector operates in a negative ion full scan mode in the 700-2000 (m/z) range.
More preferably, the chromatographic conditions of LC-MS are:
instrument: thermo Scientific UPLC-MS (equipped with DAD detector and LTQ XL mass spectrometry);
chromatographic column: acquity UPLC Oligonucleotide BEH C18 the number of columns in the Column is set, 1.7 Μm,2.1 mm. Times.50 mm, column temperature: 60 ℃;
mobile phase a:0.75% HFIP/0.0375% DIPEA (V/V)/10. Mu.M EDTA in water;
mobile phase B: methanol;
elution gradient: 1.0min phase B rises linearly from 25% to 60%, rises to 90% within 0.5min, and remains for 0.5min;
Flow rate: 0.5mL/min;
Detection wavelength: 260nm;
MS conditions: electrospray ionization (ESI) probe temperature was 300 ℃, source temperature was 350 ℃, and ESI capillary was-20 kV. The mass detector (QDa) operates in a negative ion full scan mode in the range 700-2000 (m/z). Data were analyzed by ProMass HR 2.0.0 (Novatia, pennsylvania, USA).
(4) And (3) carrying out On-DNA chemical reaction On the reference sample, carrying out pretreatment On the reaction liquid to obtain a sample liquid to be detected, mixing the sample liquid with a standard substance, carrying out LC-MS analysis under the same condition to obtain detection signal intensity, carrying the detection signal intensity into a standard working curve, calculating the quantity of substances of the reference sample in the sample liquid to be detected, obtaining the recovery rate of the reference sample, and judging the DNA damage condition of the reference sample through the recovery rate and the change of the detection signal response of the reference sample.
In order to more accurately and truly reflect the damage condition of the DNA in the On-DNA chemical reaction, the analysis method further comprises the following steps:
(5) Preparing a negative control solution containing an internal reference sample, mixing the negative control solution with a standard substance, performing LC-MS analysis under the same condition to obtain detection signal intensity, taking the detection signal intensity into a standard working curve, and calculating to obtain a negative recovery rate;
the method for quantitatively analyzing the DNA damage condition of the reference sample by calculating the recovery rate of the reference sample is as follows:
a. the final recovery rate (%) of the internal reference sample is more than or equal to the negative recovery rate (%), and the DNA label is not damaged in the On-DNA chemical reaction;
b. The final recovery (%) < negative recovery (%) of the internal reference sample indicates that the DNA tag was damaged in the On-DNA chemical reaction, and the larger the numerical difference, the more serious the DNA damage.
Meanwhile, various damage conditions generated On the DNA of the reference sample in the On-DNA chemical reaction can be qualitatively analyzed through the change of detection signal response of the reference sample (such as the charge-to-Mass ratio of molecular ion peaks in Mass, the intensity of UV peaks and the like).
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Internal reference sample preparation example
Preparation example 1
An internal reference sample S1 was prepared, and the reaction process was as follows:
Preparation of DNA-NHFmoc: 300.0nmol of Standard 1 (HP) was dissolved in deionized water to prepare a 1.0mmol/L solution (300.0. Mu.L, 300.0 nmol). 300.0. Mu.L of the above standard 1 solution was mixed with 40.0 equivalents of a DMSO solution (200.0 nmol/L) of 1- (9H-fluoren-9-ylmethyl) 4-methylmorpholine hydrochloride (DMT-MM) 1- (9H-fluoren-9-ylmethyl) ester of 5,8,11, 14-tetraoxa-2-azaheptadecane, 40.0 equivalents of an aqueous solution (200.0 mmol/L) of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM), and 250.0 equivalents of sodium tetraborate (Na 2B4O7) buffer (250.0 mmol/L) having pH=9.5, and the mixture was thoroughly mixed with a vortex shaker and reacted at 4℃for 2 hours. The reaction solution was collected by precipitation, and verified by LC-MS to give DNA-NHFmoc in a total of 240nmol (240.0. Mu.L, 240.0 nmol).
Preparation of DNA-NH 2: to the DNA-NHFmoc (240.0. Mu.L, 240.0 nmol) was added 120. Mu.L of a 10% (v/v) aqueous solution of piperidine, and the mixture was reacted at 25℃for 5 hours. The reaction solution was collected by precipitation and verified by LC-MS to give DNA-NH 2 in total 200nmol (200.0. Mu.L, 200.0nmol, MW=5184).
Preparing an internal reference sample S1: 100. Mu.L of the above DNA-NH 2 solution (100.0. Mu.L, 100.0 nmol) was mixed with 200.0 equivalents of acetonitrile solution (200.0 mmol/L) of acetyl chloride, and the mixture was thoroughly mixed with a vortex shaker and reacted at 25℃for 5 hours. The reaction solution was collected by precipitation, purified by HPLC and stored by lyophilization.
Through detection, the internal reference sample S1 has a relative molecular weight of 5226, a DNA length of 8bp and a purity of 100%.
Preparation example 2
Preparation of reference sample S2
Preparation of HP-1: 100.0nmol of Standard 1 (HP) was dissolved in deionized water to prepare a 1.0mmol/L solution (100.0. Mu.L, 100.0 nmol). 100.0. Mu.L of the above standard 1 solution was mixed with 200.0 equivalents of acetonitrile (200.0 mmol/L) of acetyl chloride, and the mixture was thoroughly mixed with a vortex shaker, and reacted at 25℃for 5 hours. The reaction solution was collected by precipitation and verified by LC-MS to give 80nmol of HP-1.
Tag 1:5'-TCCGACAGGAATG-3',5'-CGACCATTCCTGTCGGAGG-3'.
Tag 2:5'-GTCGACTACCCTTG-3',5'-TCGCAAGGGTAGT-3'.
Preparing an internal reference sample S2: after 1.1 equivalent of Tag 1 (55.0. Mu.L, 55.0 nmol) and 1.2 equivalent of Tag 2 (61.0. Mu.L, 61.0 nmol) were added to the HP-1 (50.0. Mu.L, 50.0 nmol), 40. Mu.L of 10 XDNA ligation buffer and 120U of T4 DNA ligase were added and mixed, and reacted at 25℃for 2 hours. After the reaction, the purity of the product is evaluated by LC-MS, and the product is freeze-dried and stored after HPLC purification treatment.
Through detection, the internal reference sample S2 has a relative molecular weight of 23244, a DNA length of 38bp and a purity of 95%.
Examples
Example 1
The present example provides a method for analysis of DNA damage in an On-DNA chemical reaction based On LC-MS, comprising:
1. and (3) manufacturing a standard curve:
(1) A solution of 20.0. Mu. Mol/L (1000. Mu.L, 20.0 nmol) of Standard 1 (SEQ ID NO. 15 '-/5Phos/GAGTCA/iSp9/iUniAmM/iSp 9/TGACTCCC-3') was prepared by dissolving 20.0nmol of standard 1 in deionized water. The reference sample S1 was prepared as a 20.0. Mu. Mol/L solution (1000. Mu.L, 20.0 nmol) in the same manner.
(2) Preparing 6 groups of standard solutions, wherein the molar ratio of the standard substance 1 to the internal reference sample S1 is sequentially decreased, and specifically:
a first group: taking 0.10nmol (5.0 mu L) of standard substance 1, taking 0.05nmol (2.5 mu L) of internal reference sample S1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 2;
second group: taking 0.10nmol (5.0 mu L) of standard substance 1, taking 0.10nmol (5.0 mu L) of internal reference sample S1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 1;
Third group: taking 0.10nmol (5.0 mu L) of standard substance 1, taking 0.20nmol (10.0 mu L) of internal reference sample S1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 0.5;
fourth group: taking 0.10nmol (5.0 mu L) of standard substance 1, taking 0.30nmol (15.0 mu L) of internal reference sample S1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 0.33;
Fifth group: taking 0.10nmol (5.0 mu L) of standard substance 1, taking 0.40nmol (20.0 mu L) of internal reference sample S1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 0.25;
Sixth group: standard 1 was taken at 0.10nmol (5.0 μl), reference sample S1 was taken at 0.50nmol (25.0 μl), and deionized water was used to determine the volume to 100.0 μl to prepare a solution with a molar ratio of 0.2.
(3) The prepared 6 groups of standard solutions are subjected to LC-MS detection according to the following conditions, each group of solutions is repeatedly detected for 3 times, and an average value is taken to prepare a standard curve 1.
The chromatographic conditions of LC-MS were as follows:
LC conditions:
chromatographic column: acquity UPLC Oligonucleotide BEH C18 the number of columns in the Column is set, 1.7 Μm,2.1 mm. Times.50 mm, column temperature: 60 ℃;
Mobile phase a:0.75% HFIP/0.0375% DIPEA/10. Mu.M EDTA in water;
mobile phase B: methanol;
elution gradient: 1.0min phase B rises linearly from 25% to 60%, rises to 90% within 0.5min, and remains for 0.5min;
Flow rate: 0.5mL/min;
Detection wavelength: 260nm;
MS conditions: electrospray ionization (ESI) probe temperature was 300 ℃, source temperature was 350 ℃, and ESI capillary was-20 kV. The mass detector (QDa) operates in a negative ion full scan mode in the range 700-2000 (m/z). Data were analyzed by ProMass HR 2.0.0 (Novatia, pennsylvania, USA).
Wherein, the HPLC chart of the standard 1 is shown in figure 1, and the HPLC chart of the reference sample S1 is shown in figure 2. As can be seen from the figure, under the same chromatographic conditions, the retention time of the standard 1 and the reference sample S1 are different, the separation degree is greater than 1.5, and baseline separation can be achieved.
(4) LC-MS detection results: the detection signal response of the 6 groups of standard solutions is shown in the following table 1:
Table 1.
The LC-MS signal response value in table 1 is the product of the UV response value and the Mass response value.
Both the standard 1 and the reference sample S1 in the standard solution are products with high purity and without any chemical reaction, so that the area percentage of the main peak Mass can be defaulted to 100% (Mass is less than 100% if DNA damage occurs), and the product of the UV response value and the Mass response value, namely the LC-MS signal response value, is directly shown in table 1. Some of these differences, such as the peak of the larger molecular weight 56, default to the Mass signal of the corresponding standard 1 and reference sample S1.
According to the data in the above table, regression analysis was performed with the molar ratio of standard 1 to reference sample S1 in 6 sets of standard solutions as abscissa and standard 1 (UV x Mass)/reference sample S1 (UV x Mass) as ordinate, to obtain standard working curve 1 (as shown in fig. 3): y=0.8639x+0.0047, r 2 =1. The standard curve has good linear correlation and can be used for quantitative determination of external standard.
2. Calculation of the recovery of Negative Control (NC)
100.0Nmol of reference sample S1 was dissolved in deionized water to prepare a 1.0mmol/L solution (100.0. Mu.L, 100.0 nmol). 10.0 mu L of the reference sample S1 solution was taken, 10.0 mu L of deionized water was added, and the mixture was thoroughly mixed with a vortex oscillator, and reacted at room temperature for 2 hours after the mixture was uniformly mixed. After the completion of the reaction, 6nmol was removed from the above mixture and divided into 3 parts in average, and the amount of each part of the substance was 2.0nmol as a duplicate group. To each replicate group was added 5.0mol/L sodium chloride solution in a total volume of 10%. Then, the absolute ethanol with the total volume of 2.5 times is continuously added, and after uniform shaking, the reaction solution is placed in a refrigerator at-80 ℃ for cooling overnight. After that, the supernatant was discarded by centrifugation at 4000rpm for 1 hour. The precipitate was dried at room temperature for 1 hour, and 200.0. Mu.L of deionized water was added thereto for dissolution to obtain negative control solutions (NC 1-NC 3).
20.0. Mu.L of the NC solution and 5.0. Mu.L of the standard 1 solution (0.10 nmol) were mixed, and the volume was adjusted to 100.0. Mu.L with deionized water, and 10.0. Mu.L was taken for LC-MS detection.
The detection profile is shown in fig. 4 (only one of the replicates), wherein the UV of standard 1 is 32.11% and the MASS is 100%, so that the total UV of standard 1 is uv=mass=32.11%; wherein the reference sample S1 UV accounts for 67.89% and the MASS accounts for 100%, so the total reference sample S1 accounts for UV x mass= 67.89%. Standard 1 (UV mas)/internal reference sample S1 (UV mas) was 0.473, and this value was substituted into standard curve 1 to convert to NC recovery of 92.24%.
The ratio of all repeated groups is calculated according to the method, and is substituted into a standard curve 1 to be converted to obtain the average recovery rate 94.01% +/-2.11% of NC, the precipitation loss is in a reasonable range, and meanwhile, the MASS accounts for 100% in an LC-MS spectrum, so that NC has no obvious DNA damage. The detailed recovery calculation is shown in table 2 below.
Table 2.
3. Calculation of recovery of reference sample S1 in condensation reaction (DMT-MM)
(1) Conditional feasibility verification of condensation reaction (DMT-MM)
100.0Nmol of DNA-NH 2 was dissolved in deionized water to prepare a 1.0nmol/L solution (100.0. Mu.L, 100.0 nmol). 10.0. Mu.L of the above DNA-NH 2 solution was mixed with 200.0 equivalents of FMOC-cyclopropylglycine (commercially available product, an Naiji chemical) in DMSO (200.0 mmol/L), 80.0 equivalents of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) in water (200.0 mmol/L), 250.0 equivalents of sodium tetraborate (Na 2B4O7) buffer (250.0 mmol/L) having pH=9.5, and the mixture was thoroughly mixed with a vortex shaker and reacted at room temperature for 2 hours. After the completion of the reaction, 1. Mu.L of the above mixture was taken out and subjected to precipitation treatment, followed by LC-MS detection.
The detection spectrum is shown in FIG. 5, the initial molecular weight of the known DNA-NH 2 is 5184, the molecular weight of the product is 5503, the conversion rate is 86% by LC-MS detection spectrum calculation, and the condensation reaction condition is verified to be feasible.
(2) Calculation of recovery of condensation reaction (DMT-MM)
100.0Nmol of reference sample S1 was dissolved in deionized water to prepare a 1.0mmol/L solution (100.0. Mu.L, 100.0 nmol). 10.0. Mu.L of the above reference sample S1 solution was mixed with 200.0 equivalents of FMOC-cyclopropylglycine (commercially available product) in DMSO (200.0 nmol/L), 80.0 equivalents of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) in water (200.0 mmol/L), 250.0 equivalents of sodium tetraborate (Na 2B4O7) buffer (250.0 mmol/L) having pH=9.5, and the mixture was thoroughly mixed with a vortex shaker and reacted at room temperature for 2 hours.
After the completion of the reaction, 3 parts of the above mixture was taken out in an amount of 2.0 nanomoles per part of the substance as a duplicate set with a reaction time of 2 hours.
All sample precipitation methods and sample delivery methods were identical to NC. The detection pattern is shown in fig. 6 (only one of which is repeated). The ratio of all the repeated groups was calculated, and the average recovery of the reference sample S1 condensation reaction (DMT-MM) was 88.55% + -2.02% as calculated by substituting the standard curve 1, which was less than the negative control recovery 94.65%, but no significant DNA damage was detected in the LC-MS profile, thus suggesting that slight DNA damage may occur during the On-DNA condensation reaction.
Example 2
The present example provides a method for analysis of DNA damage in an On-DNA chemical reaction based On LC-MS, comprising:
1. and (3) manufacturing a standard curve:
(1) A solution of 20.0. Mu. Mol/L (1000. Mu.L, 20.0 nmol) of Standard 1 (SEQ ID NO. 15 '-/5Phos/GAGTCA/iSp9/iUniAmM/iSp 9/TGACTCCC-3') was prepared by dissolving 20.0nmol of standard 1 in deionized water. The reference sample S2 prepared in preparation example 2 was dissolved in deionized water to prepare a solution (1000. Mu.L, 10.0 nmol) of 10.0. Mu. Mol/L.
(2) Preparing 6 groups of standard solutions, wherein the molar ratio of the standard substance 1 to the internal reference sample S2 is sequentially decreased, and specifically:
A first group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.5nmol (50 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with a molar ratio of 5;
Second group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.4nmol (40 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 4;
Third group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.3nmol (30 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 3;
Fourth group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.2nmol (20 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 2;
Fifth group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.1nmol (10 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 1;
Sixth group: taking 0.10nmol (5.0 mu L) of an internal reference sample S2, taking 0.05nmol (5.0 mu L) of a standard sample 1, and using deionized water to fix the volume to 100.0 mu L to prepare a solution with the molar ratio of 0.5;
(3) The prepared 6 groups of standard solutions are subjected to LC-MS detection according to the following conditions, each group of solutions is repeatedly detected for 3 times, and an average value is taken to prepare a standard curve 2.
LC conditions: as in example 1.
MS conditions: as in example 1.
Wherein, the HPLC chart of the standard 1 is shown in fig. 1, and the HPLC chart of the reference sample S2 is shown in fig. 7. As can be seen from the figure, under the same chromatographic conditions, the retention time of the standard 1 and the reference sample S2 are different, the separation degree is greater than 1.5, and baseline separation can be achieved.
(4) LC-MS detection results: the detection signal response of the 6 sets of standard solutions is shown in table 3 below:
Table 3.
The LC-MS signal response value in table 3 is the product of the UV response value and the Mass response value.
Standard in standard solution
The 1 main peak Mass area percentage content can be defaulted to 100%, the internal reference sample S2 main peak Mass area percentage content can be considered to be 95%, and is directly shown as the product of the UV response value and the Mass response value, namely the LC-MS signal response value in table 3. Some of these differences, such as the peak of the larger molecular weight 56, default to the Mass signal of the corresponding standard 1 and reference sample S2.
According to the data in the above table, regression analysis was performed with the molar ratio of standard 1 to reference sample S2 in 6 sets of standard solutions as abscissa and standard 1 (UV x Mass)/reference sample S2 (UV x Mass) as ordinate, to obtain standard working curve 2 (as shown in fig. 8): y=0.2306x+0.0091, and r 2 =0.9999. The standard curve has good linear correlation and can be used for quantitative determination of external standard.
2. Calculation of the recovery of Negative Control (NC)
100.0Nmol of reference sample S2 was dissolved in deionized water to prepare a 1.0mmol/L solution (100.0. Mu.L, 100.0 nmol). 10.0 mu L of the reference sample S2 solution was taken, 10.0 mu L of deionized water was added, and the mixture was thoroughly mixed with a vortex oscillator, and reacted at room temperature for 2 hours after the mixture was uniformly mixed. After the completion of the reaction, 6nmol was removed from the above mixture and divided into 3 parts in average, and the amount of each part of the substance was 2.0nmol as a duplicate group. To each replicate group was added 5.0mol/L sodium chloride solution in a total volume of 10%. Then, the absolute ethanol with the total volume of 2.5 times is continuously added, and after uniform shaking, the reaction solution is placed in a refrigerator at-80 ℃ for cooling overnight. After that, the supernatant was discarded by centrifugation at 4000rpm for 1 hour. The precipitate was dried at room temperature for 1 hour, and 200.0. Mu.L of deionized water was added thereto for dissolution to give a negative control solution (NC 4-NC 6).
20.0. Mu.L of the NC solution and 5.0. Mu.L of the standard 1 solution (0.10 nmol) were mixed, and the volume was adjusted to 100.0. Mu.L with deionized water, and 10.0. Mu.L was taken for LC-MS detection.
The detection profile is shown in fig. 9 (only one of the replicates), wherein the UV of the reference sample S2 is 70.26% and the MASS is 95.89%, so that the total UV of the reference sample S2 is uv= 67.37%; wherein the standard 1UV ratio is 29.74% and the MASS ratio is 100%, so the standard 1 total ratio is uv=mass=29.74%. Standard 1 (UV x MASS)/internal reference sample S2 (UV x MASS) was 0.441, and this value was substituted into standard curve 2 to obtain a NC recovery of 97.08% by conversion.
The ratio of all repeated groups is calculated according to the method, and is substituted into a standard curve 2 to be converted to obtain the average recovery rate of NC (95.77% +/-1.89%), the precipitation loss is within a reasonable range, and the Mass of the internal reference sample S2 is more than 95% (equivalent to the initial purity of 95%), so that NC has no obvious DNA damage. The detailed recovery calculation is shown in table 4 below.
Table 4.
3. Calculation of recovery of reference sample S2 in condensation reaction (DMT-MM)
(1) Conditional feasibility verification of condensation reaction (DMT-MM)
Has been verified in example 1.
(2) Calculation of recovery of condensation reaction (DMT-MM)
100.0Nmol of reference sample S2 was dissolved in deionized water to prepare a 1.0mmol/L solution (100.0. Mu.L, 100.0 nmol). 10.0. Mu.L of the above reference sample S2 solution was mixed with 200.0 equivalents of FMOC-cyclopropylglycine (commercially available product) in DMSO (200.0 nmol/L), 80.0 equivalents of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) in water (200.0 mmol/L) and 250.0 equivalents of sodium tetraborate (Na 2B4O7) buffer (250.0 mmol/L) having pH=9.5, and the mixture was thoroughly mixed with a vortex shaker and reacted at room temperature for 2 hours.
After the completion of the reaction, 3 parts of the above mixture was taken out in an amount of 2.0 nanomoles per part of the substance as a duplicate set with a reaction time of 2 hours.
All sample precipitation methods and sample delivery methods were identical to NC. The detection pattern is shown in fig. 10 (only one of which is repeated). The ratio of all the repeated groups was calculated, and the average recovery of the reference sample S2 condensation reaction (DMT-MM) obtained by conversion into standard curve 2 was 94.52% + -0.49%, which was slightly smaller than the recovery of the negative control, 95.77%, and no significant DNA damage was detected in the LC-MS spectrum, thereby suggesting that slight DNA damage or no DNA damage could occur during the On-DNA condensation reaction.
Example 3
Recovery calculation of reference sample S1/S2 in other On-DNA chemistry reactions:
The reference samples S1/S2 participate in 20 different types of reactions altogether, and the specific reaction condition feasibility verification and recovery rate calculation modes are as in example 1 or example 2, and are not described in detail herein, and the specific reaction conditions are shown in Table 5; recovery and DNA damage are shown in Table 6, wherein the amounts of reference samples S1/S2 used in the following reactions are as follows: S1/S2 (10.0nmol,1.0equiv,1.0mM in H 2 O), 10. Mu.L.
Table 5.
/>
/>
Table 6.
/>
/>
The reference sample S1 and the reference sample S2 were involved in 20 kinds of reactions together, and by comparing the recovery A of the reference sample S1 with the recovery B of the reference sample S2 in groups, it was found that the recovery of all the reference samples was higher than 85% in 19 kinds of reactions (except for reaction 8), and there was no significant peak shift or change in the LC-MS spectra, so that in these 19 kinds of reactions, it was considered that the reference samples were damaged by DNA only a small amount or not. Secondly, the recovery rates of the two reference samples are compared between groups, and the maximum difference of the recovery rates of the two reference samples is not more than 8% under the same reaction conditions, so that the repeatability of the result calculated by the method is good. In reaction 8, the recovery B (65.34% + -1.63%) of the reference sample S2 was much smaller than the recovery A (95.02% + -1.48%) of the reference sample S1, and in combination with the LC-MS spectrum, it was found that the depurination of the terminal base A of the reference sample S2 (terminal base was AT) (see FIG. 11, molecular weight 23109.6) was performed AT a ratio exceeding 20% of the total amount, whereas the depurination did not occur AT the terminal of the reference sample S1. Thus, the method can quantitatively analyze the recovery rate of DNA and qualitatively analyze specific DNA damage types.
To compare this method with the conventional qPCR method, we attached the reference sample S2 with the immobilized primer sequence and calculated the recovery again under the same conditions (table 6, recovery C). Some problems with the qPCR method were found by data comparison: first, qPCR method has poor reproducibility of results and large numerical fluctuation, for example, DNA damage exceeding 20% in reaction 8, but qPCR method cannot detect it. Second, the chemical residue of some reactions may severely affect the quality of qPCR, e.g., reaction 18, with abnormally high qPCR results, possibly chemical agents affecting the progress of the component reactions or fluorescence collection.
Thus, the analysis method provided by the embodiment can rapidly calculate the recovery rate of the On-DNA compound, and can verify the specific influence of tens to hundreds of chemical reactions On DNA in a short time by using only 1 internal reference sample which does not participate in the chemical reactions, which is helpful for identifying DNA damage caused by various chemical reactions in DEL library synthesis and quantitatively analyzing the recovery rate of the final product.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (7)
1. A method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS, comprising:
selecting a DNA label for carrying out On-DNA chemical reaction, and carrying out amino protection treatment On the DNA label by adopting an amino protecting group to obtain an internal reference sample;
Selecting a specific DNA fragment as a standard, wherein the standard and the reference sample are DNA or On-DNA compounds, the separation degree of a liquid chromatographic peak of the standard and the reference sample is more than 1.5, and mass spectrum peaks are not overlapped;
Preparing a group of standard solutions containing the standard substance and the internal reference sample, wherein the molar ratio of the standard substance to the internal reference sample in the standard solution is sequentially decreased; carrying out LC-MS analysis on the standard solution, carrying out regression analysis by taking the molar ratio of the standard substance to the internal reference sample as an abscissa and the ratio of the detection signal response of the LC-MS of the standard substance to the internal reference sample as an ordinate, so as to obtain a standard working curve;
Preparing a negative control solution containing the internal reference sample, mixing the negative control solution with a standard substance, performing LC-MS analysis under the same condition to obtain detection signal intensity, substituting the detection signal intensity into the standard working curve, and calculating to obtain a negative recovery rate;
and carrying out On-DNA chemical reaction On the reference sample, carrying out pretreatment On the reaction solution to obtain a sample solution to be detected, mixing the sample solution with a standard substance, carrying out LC-MS analysis under the same condition to obtain a detection signal response, carrying the detection signal response into the standard working curve, calculating the quantity of substances of the reference sample in the sample solution to be detected, obtaining the recovery rate of the reference sample, and judging the DNA damage condition of the reference sample through the recovery rate and the change of the detection signal response of the reference sample.
2. The method of claim 1, wherein the detection signal response is the peak area or peak height of the UV signal peak and Mass signal peak in the LC-MS detection profile when the standard working curve is prepared.
3. The method of claim 2, wherein the ratio of the ordinate detection signal response = standard (UV peak area x Mass peak area)/reference sample (UV peak area x Mass peak area) when creating the standard working curve.
4. The method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS according to claim 1, wherein the amino protecting group used in preparing the reference sample comprises at least one of acetyl group, phthaloyl group, p-toluenesulfonyl group, trifluoroacetyl group, nitrobenzenesulfonyl group.
5. The method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS according to claim 4, wherein the internal reference sample has the structure:
6. The method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS according to claim 1, wherein the amount of the substance of the standard in the standard solution is 1pmol-1nmol, and the amount of the substance of the reference sample is 1pmol-100nmol.
7. The method for analyzing DNA damage in an On-DNA chemical reaction based On LC-MS according to claim 1, wherein the chromatographic conditions of the LC-MS are:
Mobile phase a: is obtained by dissolving hexafluoroisopropanol, diisopropylethylamine and EDTA in water;
mobile phase B: methanol;
Flow rate: 0.3-0.8 mL/min;
Detection wavelength: 250-270 nm;
The temperature of the electrospray ionization probe is 280-320 ℃, and the source temperature is 330-370 ℃;
The ESI capillary is-18 to-22 kV;
The mass detector operates in a negative ion full scan mode in the 700-2000 (m/z) range.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310559905.1A CN116626189B (en) | 2023-05-15 | 2023-05-15 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310559905.1A CN116626189B (en) | 2023-05-15 | 2023-05-15 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116626189A CN116626189A (en) | 2023-08-22 |
| CN116626189B true CN116626189B (en) | 2024-04-30 |
Family
ID=87641081
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310559905.1A Active CN116626189B (en) | 2023-05-15 | 2023-05-15 | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116626189B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003002760A2 (en) * | 2001-06-27 | 2003-01-09 | Epigenomics Ag | Method for detecting cytosine methylation by comparatively analysing single strands of amplificates |
| CA2467629A1 (en) * | 2001-11-20 | 2003-05-30 | Oncomedx, Inc. | Methods for evaluating drug-resistance gene expression in the cancer patient |
| CN1945332A (en) * | 2006-10-01 | 2007-04-11 | 中国科学技术大学 | Method for detecting pyrimidine dipolymer content on DNA chain |
| CN101435002A (en) * | 2008-12-12 | 2009-05-20 | 深圳华大基因科技有限公司 | Method for detecting human papilloma virogene type |
| CN111965295A (en) * | 2020-07-28 | 2020-11-20 | 上海药明康德新药开发有限公司 | DNA coding shoot-head compound identification method based on LC-MS (liquid chromatography-mass spectrometry) technology verification |
| CN114764089A (en) * | 2021-01-13 | 2022-07-19 | 成都先导药物开发股份有限公司 | Method for identifying operable cut DNA coding seedling head compound |
| CN115436500A (en) * | 2021-06-04 | 2022-12-06 | 成都先导药物开发股份有限公司 | Method for identifying operable cut DNA coding seedling head compound |
| CN115436331A (en) * | 2021-06-04 | 2022-12-06 | 成都先导药物开发股份有限公司 | Method for detecting activity of DNA (deoxyribonucleic acid) coding seedling-end compound |
-
2023
- 2023-05-15 CN CN202310559905.1A patent/CN116626189B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003002760A2 (en) * | 2001-06-27 | 2003-01-09 | Epigenomics Ag | Method for detecting cytosine methylation by comparatively analysing single strands of amplificates |
| CA2467629A1 (en) * | 2001-11-20 | 2003-05-30 | Oncomedx, Inc. | Methods for evaluating drug-resistance gene expression in the cancer patient |
| CN1945332A (en) * | 2006-10-01 | 2007-04-11 | 中国科学技术大学 | Method for detecting pyrimidine dipolymer content on DNA chain |
| CN101435002A (en) * | 2008-12-12 | 2009-05-20 | 深圳华大基因科技有限公司 | Method for detecting human papilloma virogene type |
| CN111965295A (en) * | 2020-07-28 | 2020-11-20 | 上海药明康德新药开发有限公司 | DNA coding shoot-head compound identification method based on LC-MS (liquid chromatography-mass spectrometry) technology verification |
| CN114764089A (en) * | 2021-01-13 | 2022-07-19 | 成都先导药物开发股份有限公司 | Method for identifying operable cut DNA coding seedling head compound |
| CN115436500A (en) * | 2021-06-04 | 2022-12-06 | 成都先导药物开发股份有限公司 | Method for identifying operable cut DNA coding seedling head compound |
| CN115436331A (en) * | 2021-06-04 | 2022-12-06 | 成都先导药物开发股份有限公司 | Method for detecting activity of DNA (deoxyribonucleic acid) coding seedling-end compound |
Non-Patent Citations (16)
| Title |
|---|
| A solution phase platform to characterize chemical reaction compatibility with DNA-encoded chemical library synthesis;AS Ratnayake等;ACS Comb. Sci.;20190819;第21卷(第10期);第650–655页 * |
| AS Ratnayake等.A solution phase platform to characterize chemical reaction compatibility with DNA-encoded chemical library synthesis.ACS Comb. Sci. .2019,第21卷(第10期),第650–655页. * |
| B Xia等.DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry.ACS Med. Chem. Lett. .2021,第12卷(第07期),第1166–1172页. * |
| DHPLC-mtDNA控制区多态性分析系统的建立;孙宏钰, 蔡贵庆, 陆惠玲, 刘超, 陈丽娴, 李向阳, 伍新尧;法医学杂志;20051230(第04期);第265-270页 * |
| DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry;B Xia等;ACS Med. Chem. Lett.;20210603;第12卷(第07期);第1166–1172页 * |
| Lang, M等.Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria.Journal of Medicinal Chemistry.2023,第66卷(第07期),第5079-5098页. * |
| LO Hargiss 等.Reaction profiling by ultra high-pressure liquid chromatography/time-of-flight mass spectrometry in support of the synthesis of DNA-encoded libraries.Journal of Chromatography B.2014,第971卷第120-125页. * |
| N-Alkyl Linkers for DNA-Encoded Chemical Libraries;Sun, ZM等;Chemistry-An Asian Journal;20220331;第17卷(第07期);第1-10页 * |
| Reaction profiling by ultra high-pressure liquid chromatography/time-of-flight mass spectrometry in support of the synthesis of DNA-encoded libraries;LO Hargiss等;Journal of Chromatography B;20141115;第971卷;第120-125页 * |
| Sun, ZM等.N-Alkyl Linkers for DNA-Encoded Chemical Libraries.Chemistry-An Asian Journal.2022,第17卷(第07期),第1-10页. * |
| Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria;Lang, M等;Journal of Medicinal Chemistry;20230331;第66卷(第07期);第5079-5098页 * |
| 五味子蜂花粉不同萃取物对小鼠肝脏脂质过氧化及DNA氧化损伤的作用;陈思南等;食品科学;20180822;第40卷(第11期);第146-151页 * |
| 孙宏钰,蔡贵庆,陆惠玲,刘超,陈丽娴,李向阳,伍新尧.DHPLC-mtDNA控制区多态性分析系统的建立.法医学杂志.2005,(第04期),第265-270页. * |
| 徐莞媛等.脊尾白虾甘露糖结合凝集素(MBL)基因在抗镉胁迫中的生物学功能分析.渔业科学进展.2019,第41卷(第04期),第174-180页. * |
| 脊尾白虾甘露糖结合凝集素(MBL)基因在抗镉胁迫中的生物学功能分析;徐莞媛等;渔业科学进展;20190531;第41卷(第04期);第174-180页 * |
| 陈思南等.五味子蜂花粉不同萃取物对小鼠肝脏脂质过氧化及DNA氧化损伤的作用.食品科学.2018,第40卷(第11期),第146-151页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116626189A (en) | 2023-08-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11624083B2 (en) | Use of aptamers in proteomics | |
| Duncan et al. | The pros and cons of peptide-centric proteomics | |
| NO330733B1 (en) | Massemarkorer | |
| EP2124060A1 (en) | Method for high throughput peptide/protein assay generation and assays generated therewith | |
| AU2001240834A1 (en) | Mass labels | |
| CA2569379C (en) | Target-specific compomers and methods of use | |
| CN116626189B (en) | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS | |
| CN1731150B (en) | A biochip method for detecting dioxin-type chemical species | |
| CN114764089B (en) | Identification method of DNA (deoxyribonucleic acid) encoding Miao ethnic compound capable of being cut off in operation | |
| KR20190027347A (en) | Small mutation amplification and quantification method using molecular barcode and blocking oligonucleotide | |
| Li et al. | Development of novel isobaric tags enables accurate and sensitive multiplexed proteomics using complementary ions | |
| US20020106700A1 (en) | Method for analyzing proteins | |
| CN111796039B (en) | Liquid chromatography calibration method for rapidly-labeled N-glycan | |
| CN117887812B (en) | Library for high-throughput sequencing quality control, and preparation method and application thereof | |
| CN117867090B (en) | Standard substance for quantitative DNA fragmentation level and preparation method and application thereof | |
| US20220098643A1 (en) | Affinity labeling of dna-linked ligands for high throughput ligand binding assays and the uses thereof | |
| CN115420564A (en) | Homologous twin family humanized B lymphocyte cell line proteome standard substance and preparation method and application thereof | |
| CN110658163A (en) | Method for monitoring reaction in synthesis of DNA coding compound | |
| CN110791554A (en) | Quantitative method of trace DNA | |
| CN117887812A (en) | Library for high-throughput sequencing quality control, and preparation method and application thereof | |
| US20080038747A1 (en) | Method for detecting reaction of protein and sample | |
| CN117604071A (en) | Method for detecting length and distribution of plasmid DNA Poly A tail based on mass spectrum | |
| CN117165658A (en) | Kit and detection method suitable for quantification of metagenome high-throughput sequencing library | |
| US20070099221A1 (en) | Method of analyzing nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |