CN113684254A - Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry - Google Patents
Method for quantitatively detecting content of free DNA (deoxyribonucleic acid) by isotope dilution mass spectrometry Download PDFInfo
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- CN113684254A CN113684254A CN202111187105.9A CN202111187105A CN113684254A CN 113684254 A CN113684254 A CN 113684254A CN 202111187105 A CN202111187105 A CN 202111187105A CN 113684254 A CN113684254 A CN 113684254A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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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
- G01N2030/8827—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 nucleic acids
Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting the content of free DNA by an isotope dilution mass spectrometry. Firstly, a peptide nucleic acid probe is disclosed, wherein the sequence of a nucleic acid part in the peptide nucleic acid probe is shown as SEQ ID NO: 1, and the peptide partial sequence in the peptide nucleic acid probe is shown as SEQ ID NO: 2, respectively. Also discloses a method for quantitatively detecting the content of free DNA single strands by an isotope dilution mass spectrometry method, which comprises the following steps: forming free DNA-biotin-streptavidin magnetic bead complexes; adding a peptide nucleic acid probe to capture the free DNA-biotin-streptavidin magnetic bead complex; carrying out enzymolysis; redissolving; indirectly calculating the DNA content in the sample according to the peak area ratio; correcting the DNA concentration to obtain a final concentration value. The method for quantitatively detecting the content of the free DNA single strand by the isotope dilution mass spectrometry has the advantages of high accuracy, reliable result and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting the content of free DNA by an isotope dilution mass spectrometry.
Background
At present, methods for quantitatively detecting a single strand of free DNA mainly include a polymerase chain reaction method (PCR method), a molecular hybridization method, a gene chip method, a DNA binding protein method, and the like, and among them, the PCR method is most widely used. However, the PCR method requires amplification of a single strand of free DNA first and then gel imaging, which requires a lot of time and labor costs, results are susceptible to various factors, and absolute quantification is impossible.
Isotope dilution mass spectrometry is a novel direct quantitative detection method for free DNA single strands, and is a currently accepted authoritative method for accurately quantifying compounds. The method utilizes mass spectrometry to determine the peak area ratio of the mark to the non-mark object to be detected in the object to be detected added with isotope mark to indirectly calculate the concentration of the object to be detected in the sample, and corrects the DNA quantitative result according to the biotinylation efficiency, SPE recovery rate and enzymolysis efficiency, so that the detection result is accurate and reliable. Compared with other measuring methods, the method has the advantages of high specificity, high sensitivity, accurate and reliable quantitative result and the like. However, the isotope dilution mass spectrometry in the prior art still has the problems of low analysis speed and low analysis flux, and the preparation is still to be improved.
Disclosure of Invention
The invention aims to solve the technical problem of providing a safe and reliable method for measuring the content of free DNA single strands, and the method can be used for directly measuring the content of the free DNA single strands in a serum sample.
Specifically, the technical scheme of the invention is as follows:
the first aspect of the invention discloses a peptide nucleic acid probe, wherein the nucleic acid partial sequence in the peptide nucleic acid probe is shown as SEQ ID NO: 1, and the peptide partial sequence in the peptide nucleic acid probe is shown as SEQ ID NO: 2 is shown in the specification; preferably, the peptide nucleic acid probe is obtained by linking the nucleic acid moiety and the peptide moiety via polyethylene glycol.
It is understood that, in comparison to SEQ ID NO: 1 and SEQ ID NO: 2 sequences have more than 90% (e.g., 92%, 95%, 98%, etc.) homology, and functionally identical sequences are within the scope of the invention.
The second aspect of the invention discloses the application of the peptide nucleic acid probe in a method for quantitatively detecting the content of the free DNA single strand by isotope dilution mass spectrometry.
The third aspect of the invention discloses a method for quantitatively detecting the content of free DNA single strands by an isotope dilution mass spectrometry, which comprises the following steps:
s1: preparing the synthesized peptide nucleic acid probe into a solution, and preparing the synthesized isotope labeled polypeptide into a solution as an internal standard;
s2: reacting the biotinylated free DNA single strand with streptavidin magnetic beads to form a free DNA-biotin-streptavidin magnetic bead compound;
s3: adding a peptide nucleic acid probe to capture the free DNA-biotin-streptavidin magnetic bead compound, then carrying out enzymolysis on the free DNA-biotin-streptavidin magnetic bead compound, carrying out solid phase extraction on an enzymolysis product, redissolving, and carrying out quantitative detection;
s4: taking DNA standard substances with different concentrations as a horizontal coordinate, taking the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment as a vertical coordinate to establish a standard curve, and indirectly calculating the DNA content in the sample according to the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment;
s5: the final concentration value was obtained by correcting the DNA concentration.
It should be understood that the method of the present invention is not limited to the above steps, and before S1, between S1 and S2, between S2 and S3, between S3 and S4, between S4 and S5, and after S5, other additional steps may be added by those skilled in the art as needed and are within the scope of the present invention.
Preferably, the step S1 is preceded by the step S0: the synthesized free DNA single strand is prepared into a series of concentration standard solutions for establishing a standard curve.
Preferably, in S1, the sequence of the nucleic acid portion of the peptide nucleic acid probe is as set forth in SEQ ID NO: 1, the peptide part in the peptide nucleic acid probe and the sequence of the polypeptide are shown as SEQ ID NO: 2, respectively.
Preferably, in S1, the method uses13C and15n double labeling leucine in the polypeptide.
Preferably, in S3, adding sequence-modified trypsin to perform enzymolysis on the free DNA-biotin-streptavidin magnetic bead complex, performing solid-phase extraction on the enzymolysis product, drying the extract with nitrogen gas, and then redissolving the extract; and (4) carrying out HPLC-MS/MS quantitative detection on the redissolved sample.
More preferably, the reconstituted sample is quantitatively assayed using the Shimadzura X2 high performance liquid chromatography system and AB Sciex 5500 triple quadrupole LC-MS.
Preferably, the following steps are further included between S1 and S2: biotinylating the free DNA single strand and evaluating biotinylation efficiency; preferably, in S5, the enzymatic efficiency and SPE recovery rate are calculated, and the DNA concentration is corrected based on the biotinylation efficiency, enzymatic efficiency and SPE recovery rate.
The fourth aspect of the invention discloses the application of the method in the gene field.
The invention aims to solve the technical problem of providing a safe and reliable method for measuring the content of free DNA single strands, and the method can be used for directly measuring the content of the free DNA single strands in a serum sample.
In a specific embodiment of the present invention, the method for determining the content of free DNA single strands by isotope dilution mass spectrometry comprises the following steps:
(1) preparing the synthesized free DNA single strands into a series of concentration standard solutions for establishing a standard curve;
(2) preparing the synthesized peptide nucleic acid probe into a solution for capturing free DNA single strands;
(3) preparing the synthesized isotope labeling polypeptide (peptide segment in the sequence homologous peptide nucleic acid probe) into solution as an internal standard;
(4) biotinylating the free DNA single strand and evaluating biotinylation efficiency;
(5) reacting the biotinylated free DNA single strand with streptavidin magnetic beads to form a free DNA single strand-biotin-streptavidin magnetic bead compound;
(6) adding a peptide nucleic acid probe to capture a free DNA single-strand-biotin-streptavidin magnetic bead compound, and magnetically separating to remove unbound probe;
(7) adding sequence modification-level trypsin to carry out enzymolysis on the compound, carrying out solid-phase extraction on an enzymolysis product, drying an extract by using nitrogen, and redissolving the extract;
(8) carrying out HPLC-MS/MS quantitative detection on the redissolved sample;
(9) taking DNA standard substances with different concentrations as a horizontal coordinate, taking the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment as a vertical coordinate to establish a standard curve, and indirectly calculating the DNA content in the sample according to the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment;
(10) and calculating the enzymolysis efficiency and the SPE recovery rate, and correcting the DNA concentration according to the biotinylation efficiency, the enzymolysis efficiency and the SPE recovery rate.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: the amount of the synthesized free DNA single strand was 2OD, and 380. mu.L of DEPC water was added to the solution to prepare a 20. mu. mol/L free DNA single strand solution, which was then put into a 2.0mL centrifuge tube. mu.L of the DNA fragment was taken, and 900. mu.L of DEPC water was added thereto to dilute the DNA fragment to a concentration of 2. mu. mol/L as a stock solution and dispensed into 2.0mL centrifuge tubes, 100. mu.L of each tube was dispensed, and the dispensed free DNA single-stranded solution was stored in a freezer at-70 ℃. Prior to use, stock solutions were diluted in DEPC water to serial concentrations for establishing standard curves.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of (2): the peptide nucleic acid probe sequence is 5'-GTGTAATTGGTCTCCGGAAACTTTAAC-3' -PEG2-GDRALFVGEPNR, wherein 5'-gtgtaattggtctccggaaactttaac-3' (SEQ ID NO: 1) is the probe nucleic acid portion, which is complementarily paired with the free DNA sequence 5'-cacattaaccagaggcctttgaaattg-3' (SEQ ID NO: 3); GDRALFVGEPNR (SEQ ID NO: 2) is a probe peptide portion characterized by a non-human any proteinic pancreatin hydrolyzed specific peptide segment comprising the pancreatin hydrolysis site arginine R; PEG2 (polyethylene glycol) links nucleic acids to peptides. The synthesis amount of the peptide nucleic acid probe is 20nmol, 1mL of ultrapure water is accurately added to prepare a peptide nucleic acid probe solution with the concentration of 20nmol/mL (namely 20 mu mol/L), each tube is subpackaged with 100 mu L, 900 mu L of ultrapure water is added into each tube to be diluted to the concentration of 2 mu mol/L to serve as a stock solution, the stock solution is subpackaged into 2.0mL centrifuge tubes, each tube is subpackaged with 100 mu L, and the subpackaged peptide nucleic acid probe solution is stored in a refrigerator at-70 ℃. Before use, 2. mu. mol/L of a peptide nucleic acid probe stock solution was diluted to 0.05. mu. mol/L (i.e., 50 nmol/L) with ultrapure water as a working solution.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: the synthesized 1mg isotope-labeled polypeptide GDRA [ 2 ]13C6 15N]LFVGEPNR（13C and15n double-labeled leucine L) is ultrasonically dissolved by 300 mu L of acetonitrile, and then 700 mu L of ultrapure water is added to prepare an isotope labeled polypeptide stock solution with the concentration of about 1mg/mL, and the isotope labeled polypeptide stock solution is subpackaged into 2.0mL centrifuge tubes, and each tube is subpackaged by 100 mu L; adding 900 μ L of ultrapure water into 1 tube, diluting to about 0.1mg/mL, and packaging into 2.0mL centrifuge tubes, wherein each tube contains 100 μ L; adding 900 μ L of ultrapure water into 1 tube, diluting to about 0.01mg/mL, and packaging into 2.0mL centrifuge tubes, wherein each tube contains 100 μ L; taking 1 tube, adding 900 μ L ultrapure water, and diluting to about0.001mg/mL (i.e., 1 mg/L) and dispensed into 2.0mL centrifuge tubes, 100. mu.L per tube serving as stock solution, and stored in a-70 ℃ freezer. Before use, 2900. mu.L of ultrapure water was added to 100. mu.L of the 1mg/L peptide stock solution and diluted 30-fold to give an internal standard peptide fragment working solution having a concentration of about 24.93nmol/L (molecular weight of the internal standard peptide fragment 1337.5 g/moL).
The step (4) specifically comprises the following steps: biotinylating the free DNA single strand by using a Biotin 3' DNA labelling Kit, thawing all components in the Kit, removing Terminal Deoxynucleotidyl Transferase (TdT), thawing, and placing on ice; preparing TdT diluent: diluting the TdT stock solution to a working concentration of 1.5U/muL (2 muL 5 XTdT reaction buffer solution +7 muL ultrapure water +1 muL 15U/muL TdT stock solution) by using 1 XTdT reaction buffer solution, and using the TdT stock solution as a preparation; and thirdly, carrying out marking reaction, gently mixing the reaction system uniformly, and prohibiting vortex. See table 1:
TABLE 1 preparation of DNA ligation reactions
| Composition (I) | Volume of μ L | Final concentration (amount) |
| |
20 | --- |
| 5 |
10 | 1× |
| Free DNA Single Strand | 5 | 100nM |
| Biotin-11-UTP（5μM） | 5 | 0.5μM |
| TdT diluent (1.5U/. mu.L) | 5 | 0.15U/ |
| Total volume | ||
| 50 | --- |
Fourthly, incubating the reaction system at 37 ℃ for 30 min; adding 2.5 mul 0.2M EDTA to terminate the reaction; sixthly, adding 50 mu L of chloroform into each reaction tube: extracting TdT with isoamyl alcohol (volume ratio is 24: 1), quickly vortexing, then centrifuging at high speed for 1-2 mins in a microfuge tube to separate each phase, and removing and storing the upper aqueous phase for use.
DNA biotinylation efficiency: 1) configuration of RNase free 1
TE buffer solution: dilute 20 with DEPC Water
TE buffer solution, comprising the following specific steps: 10mL of 20
TE buffer +190mL DEPC Water, 1
Balancing the positive charge nylon membrane for at least 10min by using TE buffer solution;
2) the Biotin Control Oligo (1. mu.M) and the Unlabeled Control Oligo (1. mu.M) stock solutions were diluted 20-fold with TE buffer to 50nM working solution (e.g., 2. mu.L of Control Oligo + 38. mu.L of TE, pH 8.0);
3) in a microcentrifuge tube, a series of concentrations of nucleic acid standards were prepared, as shown in table 2:
TABLE 2 preparation of a series of DNA standards
4) Preparing a standard substance: adding 50 mu L of the DNA single-strand standard solution to the position A1-A5 in a 96-well plate;
5) sample preparation: in a microcentrifuge tube, 10-fold dilution of the biotinylated DNA single strands in Table 1 (e.g., 6. mu.L biotinylated DNA single strand + 54. mu.L TE, pH 8.0) with TE buffer was performed, and 50. mu.L of the biotinylated DNA single strand sample (in triplicate) was added at the A6-A8 position in a 96-well plate;
6) diluting 25 mu L (A1-A8 wells) to B1-B8 wells in a multiple ratio, wherein the diluent is TE buffer solution, and repeating the steps until reaching E1-E8 wells, and each standard substance and each sample have 5 wells;
7) the equilibrated film was placed on a clean, dry paper towel. Excess buffer was blotted dry, but to avoid drying the membrane;
8) 2 mul of sample and standard were pipetted onto a positively charged nylon membrane and allowed to absorb;
9) UV cross-linked film: crosslinking the membrane under a 312nm UV lamp for 10-15min, and placing the membrane in a superclean bench to irradiate the UV lamp;
10) blocking Buffer and 4 were placed in a 37-50 ℃ water bath
Wash Buffer (Wash Buffer) was gently rewarmed until all particles were dissolved. The above Buffer can be normally used between room temperature and 50 ℃, and the Substrate Equilibration Buffer can be normally used between 4 ℃ and room temperature;
11) sealing the membrane: placing the membrane into a clean container, placing the container on a shaking table, adding 16mL Blocking Buffer (confining liquid) into the container, and incubating for 15min in a gentle shaking way;
12) prepare conjugate/blocking buffer solution: add 50 μ L Stabilized Streptavidin-Horseradish Peroxidase Conjugate to 16mL Blocking Buffer (1: 300 dilution);
13) pouring out the blocking buffer (blocking solution) of the previous blocking membrane, adding 16mL of the conjugate/blocking buffer solution prepared in the step 3, and incubating for 15min in a gentle swing manner;
14) preparation 1
wash solution: adding 40mL of 4
Wash Buffer to 120mL of ultrapure water;
15) transfer the membrane to a new container using 20mL of 1
wash solution simple flushing;
16) the membrane was placed in a clean container using 20mL of 1
washing the membrane for 5min for 4 times, wherein the washing mode is that the membrane is gently swung on a shaking table;
17) the membrane was transferred to a new vessel, 30mL of Substrate Equisibration Buffer was added and incubated gently with rocking for 5 min;
18) preparing a chemiluminiscent Substrate working solution: 6mL of Luminol/Enhancer Solution (Luminol/Enhancer Solution) was added to 6mL of Stable Peroxide Solution (stabilized Peroxide Solution). Note that: exposure to sunlight or any intense light can damage the working fluid. The working solution is not damaged after being exposed to laboratory lamplight for a short time;
19) the excess Buffer was removed from the membrane by lifting it from the Substrate Equilibration Buffer and placing the edge on a paper towel. Placing the film in a clean container or on a plastic preservative film with a smooth surface;
20) pouring the working solution of Chemiluminescent Substrate on the membrane to completely cover the membrane, and standing and incubating for 5 min;
21) the membrane was removed from the chemiluminscent Substrate working solution and the edges placed on a paper towel for 2-5 seconds to remove excess buffer, taking care not to dry the membrane;
22) the film was wrapped with cling film, taking care to avoid air bubbles and wrinkles. Placing the membrane in BIO-RAD ChemiDocTMSignal intensity was detected in the XRS + Imaging System. Taking the five standard biotinylation efficiencies (100%, 75%, 50%, 25% and 0%) in the table 2 as abscissa, taking the saturation signal intensity corresponding to each standard as ordinate to establish a standard curve, and then substituting the sample saturation biotinylation signal intensity into the equation to obtain the sample biotinylation efficiency.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of: placing a magnetic bead bottle on a vortex oscillator for 20s, and oscillating to resuspend magnetic beads; transferring 20 mu L (10 mg/mL, which is equivalent to 0.2 mg) of magnetic beads into a 2.0mL new centrifuge tube by using a pipettor, placing the centrifuge tube on a magnetic separator, standing for 1min (the operation is hereinafter referred to as magnetic separation), sucking supernatant by using the pipettor, and taking the centrifuge tube off the magnetic separator; ③ adding 1mL of Buffer I (10 mM Tris-HCl (pH 7.5), 1mM EDTA, 1M NaCl, 0.01-0.1% Tween-20) into a centrifuge tube, covering a centrifuge tube cover, fully oscillating the resuspension magnetic beads, then carrying out magnetic separation, and removing supernatant; fourthly, repeating the step 3 once; adding 500 μ L of biotinylated nucleic acid diluted with Buffer I (to make the concentration of magnetic beads be 0.4 mg/mL), fully oscillating the resuspended magnetic beads, placing the centrifuge tube on a rotary mixer, and rotationally mixing at room temperature for 30 min; magnetic separation, transferring the supernatant to a new centrifuge tube;
and (4) washing the magnetic beads for 3 times according to the method in the step (3) to obtain the DNA-biotin-streptavidin magnetic bead compound.
The isotope dilution of the inventionThe method for determining the content of the free DNA single strand by using the mass spectrometry, wherein the step (6) specifically comprises the following steps: 1mL of hybridization buffer (10 mM Tris, 100 mM KCl, 1mM MgCl) was added to each of the free DNA single strand-biotin-streptavidin magnetic bead complexes2pH 7.4) and 20. mu.L of peptide nucleic acid probe solution (0.05. mu. mol/L), and placed in a hybridization instrument for hybridization at 65 ℃ for 16h to form a peptide nucleic acid probe-DNA-biotin-streptavidin magnetic bead complex. And then washing the complex with PBS and carrying out magnetic separation, removing the supernatant, repeating for many times, and removing the unbound peptide nucleic acid probe as much as possible to obtain the pure peptide nucleic acid probe-DNA single-strand-biotin-streptavidin magnetic bead complex.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry, disclosed by the invention, comprises the following steps of (7): resuspending the peptide nucleic acid probe-DNA single-chain-biotin-streptavidin magnetic bead compound by using 950 mu L of ultrapure water; adding 50 mu L isotope labeled polypeptide working solution (24.93 nmol/L); ③ adding 10 mu L of pancreatin (0.04 mu g/mu L), and standing at 37 ℃ for enzymolysis for 4 h. Since the pancreatin can specifically hydrolyze the peptide bond formed by arginine R and other amino acid hydroxyl groups, the pancreatin can hydrolyze the complex into 5'-GTGTAATTGGTCTCCGGAAACTTTAAC-3' -PEG2-GDR and ALFVGEPNR, and hydrolyze the isotope labeled polypeptide into GDR and A [, ]13C6 15N]LFVGEPNR; fourthly, the enzymolysis product is purified and enriched by Sep-pak C18 cartridges (60mg, 3mL) solid phase extraction column to ALFVGEPNR and A [, ]13C6 15N]LFVGEPNR. The Sep-pak C18 cartridges were first activated and equilibrated with 3mL of methanol and 3mL of ultrapure water, respectively, then the enzymatic hydrolysate was added, the cartridges were washed with 2mL of ultrapure water, and finally ALFVGEPNR and A were extracted with 1mL of a methanol solution containing 0.2% formic acid13C6 15N]LFVGEPNR; drying the extract by using nitrogen; sixthly, 400 mu L of 0.1 percent formic acid aqueous solution is added to redissolve ALFVGEPNR and A after blow drying13C6 15N]LFVGEPNR polypeptide mixtures.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry of the invention comprises the following step (8)The method comprises the following steps: using the Shimadzu Nexera X2 high performance liquid chromatography system and AB Sciex 5500 triple quadrupole LC-MS with Symmetry ShieldTMRP18 (2.1X 150mm, 3.5 μm) column was chromatographed at 40 ℃. The mobile phase is as follows: a: an aqueous solution containing 0.1% formic acid; b: 0.1% formic acid in methanol; the flow rate is 0.2 mL/min; the sample volume is 10 mu L; elution was performed according to the following gradient: b5% (0-0.1 min) → B35% (0.1-3min) → B80% (3-3.1min) → B80% (3.1-5min) → B5% (5-5.1min) → B5% (5.1-8 min). Using a positive ion Multiple Reaction Monitoring (MRM) mode to monitor ALFVGEPNR and A [, ]13C6 15N]Signal strength of two LFVGEPNR polypeptides. The ion source of the mass spectrum system is an electrospray ionization source (ESI), and the spray voltage is 5500V; atomization pressure (Nebulizer pressure) 55 psi; the atomization temperature is 550 ℃; atomizing airflow is 10L/min; q1 and Q3 mass selectors each set at unit mass resolution; ions used for quantitative analysis are m/z 501.0 → 571.4/501.0 → 670.5 (ALFVGEPNR) and m/z 505.5 → 572.2/505.5 → 671.4 (A [ 2 ]13C6 15N]LFVGEPNR); the collision energy CE is 23 eV; the declustering voltage DP was 100V. All data were collected and processed by AB Sciex multisant workstation software (version 3.0.2).
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry comprises the following steps of (9): firstly, a standard curve is established, and a free DNA single-strand stock solution with the concentration of 2 mu mol/L is diluted into the following series of concentrations by DEPC water: 200nmol/L, 100nmol/L, 50nmol/L, 10nmol/L and 1nmol/L, then operating according to the specific steps of the steps (4) - (8), and adopting Shimadzu Nexera X2 high performance liquid chromatography and AB Sciex 5500 triple quadrupole liquid chromatography to extract the polypeptide product ALFVGEPNR and the isotopically labeled polypeptide A [ 2 ]13C6 15N]LFVGEPNR peak area was measured with the concentration of free DNA single strand as abscissa (x) The peak area ratio of the two polypeptides (A), (B), (C), (D), (E), (D), (E), (B), (E) and (B), (E), (y) Establishing a standard curve for the ordinate, the equation of the curve beingy=0.002x+0.4173（R 2= 0.956); ② when quantifying free DNA single strand, the peptide nucleic acid ALFVGEPNR and the like are extractedThe site-directed polypeptide A [ alpha ], [ beta ] -a13C6 15N]And substituting the LFVGEPNR peak area ratio into the curve equation to calculate the content of the free DNA single strand in the sample.
The method for determining the content of the free DNA single strand by the isotope dilution mass spectrometry comprises the following steps of (10): firstly, evaluating the recovery rate of SPE, and taking 400 mu L of isotope labeled peptide (24.93 nmol/L) as a control group; another 400. mu.L isotope labeled peptide (24.93 nmol/L) is taken to be subjected to solid phase extraction by Sep-pak C18 cartridges, the process is the same as the step (8), then 400. mu.L 0.1% formic acid aqueous solution is added for redissolution (experimental group), and HPLC-MS/MS detection is respectively carried out on the samples of the control group and the experimental group. The ion pair for quantitative analysis was m/z 669.1 → 572.2/669.1 → 671.4 (GDRA [ 2 ]13C6 15N]LFVGEPNR); the collision energy CE is 44 eV; the declustering voltage DP was 100V. All data were collected and processed by AB Sciex multisant workstation software (version 3.0.2). Calculating SPE recovery rate by comparing the peak area ratio of the two, namely SPE recovery rate (%) = (peak area of post-SPE internal standard peptide/peak area of non-SPE internal standard peptide) × 100%; ② evaluating the enzymolysis efficiency, taking 1mL of internal standard peptide working solution (24.93 nmol/L), adding 10 μ L pancreatin (0.04 μ g/μ L), placing at 37 ℃ for enzymolysis for 4h, then performing SPE, blow-drying and redissolution according to the above steps, then performing HPLC-MS/MS detection, wherein the ion pairs for quantitative analysis are m/z 669.1 → 572.2/669.1 → 671.4 (GDRA [, ])13C6 15N]LFVGEPNR, original peptide fragment) and m/z 505.5 → 572.2/505.5 → 671.4 (A2 [ ]13C6 15N]LFVGEPNR, enzymatically cleaved peptide fragment). Enzymolysis efficiency (%) = [ enzymolysis peptide peak area/(enzymolysis peptide peak area + original peptide peak area)]The SPE recovery rate is multiplied by 100 percent;
and correcting the DNA concentration according to biotinylation efficiency, SPE recovery rate and enzymolysis efficiency.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the method has the following advantages:
the method directly quantifies the free DNA single chains in the sample based on the isotope dilution mass spectrometry technology, and quantifies the biotinylation efficiency, the SPE recovery rate and the enzymolysis efficiency, so that the DNA quantification result is accurate and reliable; the DNA single strand does not need to be amplified in the determination process, so that the influence of the amplification efficiency is avoided; and the Shimadzu Nexera X2 high performance liquid chromatography system and the AB Sciex 5500 triple quadrupole LC-MS can accommodate about 100 samples for simultaneous detection, and the analysis speed and the analysis flux are remarkably superior to those of the common method.
Drawings
FIG. 1 is a flow chart of an experiment in an embodiment of the present invention;
FIG. 2 is a graph showing a standard curve of the biotinylation efficiency of DNA in an example of the present invention;
FIG. 3 is HPLC-MS/MS chromatogram of 24.93nmol/L internal standard peptide GDRA [13C615N ] LFVGEPNR, HPLC-MS/MS chromatogram after enzymolysis of 50nmol/L peptide nucleic acid probe 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG 2-G-DRALFVGEPNR and 24.93nmol/L internal standard peptide GDRA [13C615N ] LFVGEPNR in the example of the present invention;
FIG. 4 is a standard curve diagram established by taking DNA standards with different concentrations as abscissa and taking peak area ratio of peptide nucleic acid and isotope-labeled enzymolysis peptide fragment as ordinate in the embodiment of the present invention;
FIG. 5 is a graph of enzymatic hydrolysis efficiency in an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Instruments and reagents: AB Sciex 5500 triple quadrupole mass spectrometer (Applied biosystems, USA) equipped with electrospray ion source; shimadzu Nexera X2 ultra high performance liquid chromatograph (Japan, shimadzu corporation) was equipped with a binary high-pressure pump, an autosampler, and a column oven; ABN2ZA Nitrogen gas generator (PEAK, Inc., USA); SymmetryShieldTMRP18 chromatography column (2.1X 150mm, 3.5 μm) (Waters corporation, USA) AllegraTM64R Centrifuge desktop high speed refrigerated Centrifuge (BECKMAN COULTER, USA); an Eppendorf pipettor (Eppendorf, germany); G560E vortex mixer (Scientific Industries, USA); n2 evaluation System NV-15G Nitrogen blower (Agela Technologies, Germany); eppendorf ThermoMixer C hybrid instrument (U.S. A, Eppendorf corporation); rotary shaker QB-208 type (Haiman, Tubeier instruments, Inc., China); a DKB-501S constant temperature water bath (China, Shanghai sperm macro experimental facilities, Inc.); CSB-10BT ultrasonic cleaner (Hangzhou Aipu Instrument, Inc., China); Milli-Q ultrapure water meter (Merck Millipore, Germany). Chromatographically pure methanol and acetonitrile were purchased from j.t. Baker corporation (usa); NH (NH)4HCO3And Formic Acid (FA) from Fluke corporation (usa); PBS was purchased from Sigma-Aldrich (USA); sep-pak®C18 (60mg, 3mL) solid phase extraction column was purchased from Waters corporation (USA); sequence-modified pancreatic enzymes were purchased from Promega (usa); ultrapure water was prepared by the laboratory Milli-Q ultrapure water meter; biotin 3' DNA Labeling Kit was purchased from Thermo Fisher Scientific, Inc. (USA); streptavidin magnetic beads were purchased from coypu biomedical engineering ltd (china); peptide nucleic acid probes were synthesized by Hangzhou Taihe Biotechnology, Inc. (China); isotope-labeled polypeptides were synthesized by Shanghai Qiaozhao Biotechnology, Inc. (China); free DNA single-stranded nucleic acid was synthesized by Shanghai Bioengineering Co., Ltd. (China).
Example 1
In this embodiment, an experimental flowchart of a method for measuring a single-stranded free DNA in serum by using isotope dilution ultra-high performance liquid chromatography-mass spectrometry is shown in fig. 1, and includes the following steps:
(1) preparing a free DNA single-stranded nucleic acid single-stranded solution, a peptide nucleic acid probe solution and an isotope labeled polypeptide internal standard solution:
the amount of the synthesized free DNA single strand was 2OD, and 380. mu.L of DEPC water was added to the solution to prepare a 20. mu. mol/L free DNA single strand solution, which was then put into a 2.0mL centrifuge tube. mu.L of the DNA fragment was taken, and 900. mu.L of DEPC water was added thereto to dilute the DNA fragment to a concentration of 2. mu. mol/L as a stock solution and dispensed into 2.0mL centrifuge tubes, 100. mu.L of each tube was dispensed, and the dispensed free DNA single-stranded solution was stored in a freezer at-70 ℃. Before use, the stock solution was diluted with DEPC water to a range of concentrations for establishing a standard curve;
the synthesis amount of the peptide nucleic acid probe is 20nmol, 1mL of ultrapure water is accurately added to prepare a peptide nucleic acid probe solution with the concentration of 20nmol/mL (namely 20 mu mol/L), each tube is subpackaged with 100 mu L, 900 mu L of ultrapure water is added into each tube to be diluted to the concentration of 2 mu mol/L to serve as a stock solution, the stock solution is subpackaged into 2.0mL centrifuge tubes, each tube is subpackaged with 100 mu L, and the subpackaged peptide nucleic acid probe solution is stored in a refrigerator at-70 ℃. Before use, 2. mu. mol/L peptide nucleic acid probe stock solution was diluted to 0.05. mu. mol/L (i.e., 50 nmol/L) with ultrapure water as a working solution;
isotope labeled polypeptide GDRA [ 2 ]13C6 15N]The synthesis amount of LFVGEPNR is 1mg, 300 mu L of acetonitrile is firstly used for ultrasonic dissolution, then 700 mu L of ultrapure water is added to prepare an isotope labeled polypeptide stock solution with the concentration of about 1mg/mL, the isotope labeled polypeptide stock solution is subpackaged into 2.0mL centrifuge tubes, and each tube is subpackaged with 100 mu L; adding 900 μ L of ultrapure water into 1 tube, diluting to about 0.1mg/mL, and packaging into 2.0mL centrifuge tubes, wherein each tube contains 100 μ L; adding 900 μ L of ultrapure water into 1 tube, diluting to about 0.01mg/mL, and packaging into 2.0mL centrifuge tubes, wherein each tube contains 100 μ L; the sample was diluted to about 0.001mg/mL (i.e., 1 mg/L) in 900. mu.L of ultrapure water in 1 tube, and the diluted sample was dispensed into 2.0mL centrifuge tubes, and 100. mu.L of each tube was dispensed as a stock solution and stored in a refrigerator at-70 ℃. Before use, 2900 μ L of ultrapure water is added into 100 μ L of 1mg/L peptide fragment stock solution to dilute the stock solution by 30 times, so as to obtain an internal standard peptide fragment working solution with the concentration of about 24.93nmol/L (the molecular weight of the internal standard peptide fragment is 1337.5 g/moL);
free DNA single-stranded stock solution at a concentration of 2. mu. mol/L was diluted with DEPC water to the following series of concentrations: 200nmol/L, 100nmol/L, 50nmol/L, 10nmol/L and 1 nmol/L;
diluting the peptide nucleic acid probe stock solution to 50nmol/L by using ultrapure water as a peptide nucleic acid probe working solution; diluting the isotope labeling polypeptide stock solution to about 24.93nmol/L by using ultrapure water as isotope labeling internal standard polypeptide working solution;
HPLC-MS/MS chromatogram of 24.93nmol/L internal standard peptide GDRA [13C615N ] LFVGEPNR, HPLC-MS/MS chromatogram after enzymolysis of 50nmol/L peptide nucleic acid probe 5'-CTAAACGGAACCACTAGTGACTTGA-3' -PEG 2-G-DRALFVGEPNR and 24.93nmol/L internal standard peptide GDRA [13C615N ] LFVGEPNR are shown in figure 3.
(2) Establishing a free DNA single-strand standard curve: and precisely absorbing 5 mu L of free DNA single-stranded solution with each concentration respectively, biotinylating the free DNA single-stranded solution according to the operation steps of the Biotin 3' DNA Labeling Kit specification, and calculating biotinylation efficiency. And (3) reacting all the biotinylated free DNA single strands with 50 mu L of the treated streptavidin magnetic bead, placing the mixture on a rotary shaker, and rotationally mixing the mixture at room temperature for 30min to form a free DNA single strand-biotin-streptavidin magnetic bead complex. Adding 1mL of hybridization buffer solution into the free DNA single-chain-biotin-streptavidin magnetic bead complex, then adding 30 μ L of peptide nucleic acid probe solution (50 nmol/L), fully mixing uniformly, placing in a hybridization instrument for hybridization, carrying out 16h at 65 ℃ to capture the free DNA single-chain-biotin-streptavidin magnetic bead complex to form the peptide nucleic acid probe-free DNA single-chain-biotin-streptavidin magnetic bead complex, then repeatedly washing the complex with PBS and carrying out magnetic separation, removing the unbound peptide nucleic acid probe as much as possible, and obtaining the pure peptide nucleic acid probe-free DNA single-chain-biotin-streptavidin magnetic bead complex. The peptide nucleic acid probe-free DNA single strand-biotin-streptavidin magnetic bead complex was redissolved with 950. mu.L of ultrapure water, 50. mu.L of isotope-labeled polypeptide solution (24.93 nmol/L) was added, 10. mu.L of pancreatin (0.04. mu.g/. mu.L) was added, and the mixture was placed in a hybridization apparatus for enzymatic hydrolysis at 37 ℃ for 4 hours. The enzymolysis product is treated with Sep-pak®The C18 cartridge solid phase extraction column is purified to enrich ALFVGEPNR and A [, ]13C6 15N]LFVGEPNR, blow-dried and redissolved with 400. mu.L of 0.1% formic acid methanol solution;
under the same preset condition of the ultra performance liquid chromatography mass spectrum, respectively injecting a series of re-dissolved polypeptide and isotope labeled polypeptide samples with different known concentrations, of which the volume is 10 mu L, into an ultra performance liquid chromatography mass spectrum combination instrument to obtain a series of chromatograms of free DNA single-strand standard curve working solutions with different known concentrations;
the peptide segment ALFVGEPNR is enzymolyzed by the re-dissolved PNA and the internal standard peptide segment A13C6 15N]The ratio of peak areas of LFVGEPNR chromatographic peak is used as a vertical coordinate, the concentrations of different free DNA single-strand working solutions are used as a horizontal coordinate, a free DNA single-strand standard curve is established, and a regression equation isy=0.002x+0.4173（R 2= 0.956), the results are shown in table 4 and fig. 4;
TABLE 4 peak area ratios of peptide fragments
| Concentration of | Peak area of peptide by PNA enzymolysis | Peak area of internal standard zymolytic peptide | Peak area ratio |
| 1nM | 3.65E+03 | 9.61E+03 | 0.38 |
| 10nM | 9.46E+03 | 2.05E+04 | 0.46 |
| 50nM | 7.86E+03 | 1.55E+04 | 0.51 |
| 100nM | 9.78E+03 | 1.48E+04 | 0.66 |
| 200nM | 1.44E+04 | 1.82E+04 | 0.79 |
Chromatographic conditions are as follows: a chromatographic column: SymmetryShieldTMRP18 (2.1X 150mm, 3.5 μm) chromatography column; mobile phase: a: an aqueous solution containing 0.1% formic acid; b: acetonitrile solution containing 0.1% formic acid, gradient elution, elution procedure: b5% (0-0.1 min) → B35% (0.1-3min) → B80% (3-3.1min) → B80% (3.1-5min) → B5% (5-5.1min) → B5% (5.1-8 min); flow rate: 0.2 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution;
mass spectrum conditions: an ion source: an electrospray ion source; scanning mode: a positive ion mode; capillary voltage: 5500V; ion source temperature: 550 ℃; ion source atomization gas: 55 psi; heating auxiliary gas by an ion source: 60 psi; air curtain air: 30 psi; collision gas: 4 psi; the gas is nitrogen; scanning mode: multiple reaction monitoring, the multiple reaction monitoring conditions are shown in table 5 below:
TABLE 5 multiple reaction monitoring conditions for peptide fragments
(3) Determination of the concentration of free DNA single strands in the sample: subjecting the polypeptide ALFVGEPNR and the isotope labeled polypeptide A in the sample solution13C6 15N]Substituting the ratio of the LFVGEPNR peak area into the established standard curve equation, and calculating the concentration of the free DNA single strand in the sample;
(4) calculating biotinylation efficiency:
establishment of biotinylation standard curve: the five standard biotinylation efficiencies (100%, 75%, 50%, 25% and 0%) in the above table 2 were used as abscissa, the signal intensity corresponding to each standard was used as ordinate to establish a standard curve, and the regression equation wasy=0.6064x-2.38（R 2= 0.9945), see fig. 2. And substituting the biotinylation signal intensity of the biotinylation sample into the equation to obtain the biotinylation efficiency of the sample.
(5) And (3) SPE recovery rate calculation:
SPE recovery (%) = (peak area of post-SPE internal standard peptide/peak area of non-SPE internal standard peptide) × 100%;
two tubes are respectively taken, 1mL of the 25nmol/L internal standard peptide GDRA 213C6 15N]LFVGEPNR working solution, wherein one tube carries out solid phase extraction according to the following steps: the Sep-pak C18 cartridges are first activated and equilibrated with 3mL of methanol and 3mL of ultrapure water, respectively, followed by addition of 1mL of 25nmol/L internal standard peptide GDRA [, [ solution of13C6 15N]LFVGEPNR working solution, then 2mL of ultrapure water is used for cleaning the column, then 1mL of methanol solution containing 0.2% formic acid is used for extraction, finally the extract is dried by nitrogen, and after drying, 1mL of 0.1% formic acid aqueous solution is added for redissolution. And (3) respectively carrying out HPLC-MS/MS detection on the two tubes of internal standard peptide working solutions without SPE and SPE, detecting each tube for 3 times, calculating the SPE recovery rate by calculating the peak area ratio of the two tubes, wherein the SPE recovery rate is 94.9%, and the results are shown in Table 6.
TABLE 6 SPE recovery
(6) Calculating enzymolysis efficiency:
enzymolysis efficiency (%) = [ enzymolysis peptide peak area/(enzymolysis peptide peak area + original peptide peak area) ]/SPE recovery (%) x 100%;
taking 1mL of internal standard peptide fragment working solution (24.93 nmol/L), adding 10 μ L of pancreatin (0.04 μ g/μ L), performing enzymolysis at 37 ℃ for 4h, performing SPE, blow-drying and redissolving according to the steps, and performing HPLC-MS/MS detection (n = 3) for quantitative fractionationThe ion pair to be eluted is m/z 669.1 → 572.2/669.1 → 671.4 (GDRA [ 2 ]13C6 15N]LFVGEPNR, original peptide fragment) and m/z 505.5 → 572.2/505.5 → 671.4 (A2 [ ]13C6 15N]LFVGEPNR, enzymatic peptide fragment), the enzymatic efficiency was calculated according to the above formula, the enzymatic efficiency was 77.0%, and the results are shown in table 7 and fig. 5.
TABLE 7 enzymatic efficiency
The results show that the isotope dilution ultra-liquid chromatography-mass spectrometry combined method for measuring the free DNA single strand constructed in the embodiment is simple, rapid, high in specificity and high in accuracy.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Sequence listing
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<120> method for quantitatively detecting content of free DNA by isotope dilution mass spectrometry
<150> 2020111229005
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Claims (10)
1. A peptide nucleic acid probe, wherein the sequence of a nucleic acid part in the peptide nucleic acid probe is shown as SEQ ID NO: 1, and the peptide partial sequence in the peptide nucleic acid probe is shown as SEQ ID NO: 2 is shown in the specification; preferably, the peptide nucleic acid probe is obtained by linking the nucleic acid moiety and the peptide moiety via polyethylene glycol.
2. The peptide nucleic acid probe of claim 1, in a method for quantitative determination of free DNA single strand content by isotope dilution mass spectrometry.
3. A method for quantitatively detecting the content of free DNA single strands by an isotope dilution mass spectrometry method is characterized by comprising the following steps:
s1: preparing the synthesized peptide nucleic acid probe into a solution, and preparing the synthesized isotope labeled polypeptide into a solution as an internal standard;
s2: reacting the biotinylated free DNA single strand with streptavidin magnetic beads to form a free DNA-biotin-streptavidin magnetic bead compound;
s3: adding a peptide nucleic acid probe to capture the free DNA-biotin-streptavidin magnetic bead compound, then carrying out enzymolysis on the free DNA-biotin-streptavidin magnetic bead compound, carrying out solid phase extraction on an enzymolysis product, redissolving, and carrying out quantitative detection;
s4: taking DNA standard substances with different concentrations as a horizontal coordinate, taking the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment as a vertical coordinate to establish a standard curve, and indirectly calculating the DNA content in the sample according to the peak area ratio of the peptide nucleic acid enzymolysis peptide segment and the isotope labeled enzymolysis peptide segment;
s5: the final concentration value was obtained by correcting the DNA concentration.
4. The method according to claim 3, wherein the step S1 is preceded by the step S0: the synthesized free DNA single strand is prepared into a series of concentration standard solutions for establishing a standard curve.
5. The method according to claim 3, wherein in S1, the sequence of the nucleic acid portion in the peptide nucleic acid probe is as set forth in SEQ ID NO: 1, the peptide part in the peptide nucleic acid probe and the sequence of the polypeptide are shown as SEQ ID NO: 2, respectively.
6. The method according to claim 3, wherein in S1, the method is adopted13C and15n double labeling leucine in the polypeptide.
7. The method according to claim 3, wherein in S3, the free DNA-biotin-streptavidin magnetic bead complex is subjected to enzymolysis by adding sequence modification-grade trypsin, the enzymolysis product is subjected to solid phase extraction, the extract is dried by nitrogen gas, and then the extract is redissolved; and (4) carrying out HPLC-MS/MS quantitative detection on the redissolved sample.
8. The method according to claim 7, wherein the reconstituted sample is quantitatively assayed using the Shimadzu Nexera X2 high performance liquid chromatography system and AB Sciex 5500 triple quadrupole LC Mass spectrometer.
9. The method of claim 3, further comprising, between S1 and S2, the steps of: biotinylating the free DNA single strand and evaluating biotinylation efficiency; preferably, in S5, the enzymatic efficiency and SPE recovery rate are calculated, and the DNA concentration is corrected based on the biotinylation efficiency, enzymatic efficiency and SPE recovery rate.
10. Use of the method according to claims 3-9 in the genetic field.
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