CN113655223A - Multiplex amino acid quantitative method and kit development - Google Patents

Multiplex amino acid quantitative method and kit development Download PDF

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CN113655223A
CN113655223A CN202110710806.XA CN202110710806A CN113655223A CN 113655223 A CN113655223 A CN 113655223A CN 202110710806 A CN202110710806 A CN 202110710806A CN 113655223 A CN113655223 A CN 113655223A
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amino acid
kit
acid
amino acids
standard amino
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周愿
洪聪聪
田璐
吴祥佳
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Xuzhou Medical University
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    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

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Abstract

The invention relates to a novel Multiple Amino Acid Quantification (MAAQ) method and a related kit. The technical scheme of the invention is as follows: the amino acids are chemically labeled by organic combination of formaldehyde and sodium cyanoborohydride and their respective isotopic forms, so that the amino acids are provided with different mass labels, and quantitative analysis of the amino acids in multiple samples is realized by selective ion Scanning (SIM), Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM) or non-data dependent acquisition (DIA). The method and the kit have the advantages of high quantitative flux, low cost, high accuracy and the like, and the kit can be used for greatly facilitating the quantification of the amino acid.

Description

Multiplex amino acid quantitative method and kit development
Technical Field
The invention designs an amino acid quantitative method based on mass spectrum technology, which can realize large-scale, high-accuracy and high-precision quantification of amino acid samples and has the advantages of low price, simple method marking, high marking efficiency, no side reaction and the like. The method can be used for clinical diagnosis and quantitative analysis of amino acids contained in food samples.
Background
Amino acids are important components of life and are constituent units of proteins. There are more than 40 kinds of important amino acids in human body, including 20 kinds of protein amino acids and some amino acids with important functions, which are collectively called "full spectrum amino acids". Specifically, the compound comprises arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, gamma-aminobutyric acid, glycine, serine, taurine, tyrosine, alpha-aminocaproic acid, asparagine, aspartic acid, citrulline, glutamic acid, glutamine, ornithine, cysteine, cystathionine, homocysteine, alpha-aminon-butyric acid, alanine, anserine, beta-alanine, beta-aminoisobutyric acid, carnosine, ethanolamine, delta-hydroxylysine, hydroxylated proline, 1-methylhistidine, 3-methylhistidine, phosphoethanolamine, phosphoserine, proline, sarcosine, argininosuccinic acid and hydroxylated citrulline. The full spectrum of amino acids is related to many diseases, such as metabolic diseases, tumors, immunity, cardiovascular and cerebrovascular diseases, nervous system diseases and the like, and is also related to skeletal development, hormone secretion and the like of children. In addition, the content of amino acids is also required to be tested in food testing to determine the efficacy of the food.
The existing quantitative method mainly combines a derivation method with a liquid chromatography, a gas chromatography-mass spectrometry and a liquid chromatography-mass spectrometry combined method. The liquid chromatography-mass spectrometry combined method can simultaneously realize qualitative and quantitative determination due to simple sample treatment, and is widely applied. The application of the liquid chromatography-mass spectrometry method is based on a stable isotope labeling addition method, and stable isotopes can be introduced by adding amino acids containing 13C, 15N or 2H elements in the sample pretreatment process and carrying out chemical labeling by using reagents containing the elements. However, the detection cost and the throughput of the method are low, so that the clinical application of the method is limited.
Disclosure of Invention
Therefore, a novel labeling method is designed, organic combination is carried out by using formaldehyde, sodium cyanoborohydride and isotope forms thereof, as shown in table 1, the simultaneous quantitative analysis of the highest eight samples is realized, and the analysis cost of a single sample is lower than 1/10 of the existing analysis method.
TABLE 1 summary of the combined labeling of formaldehyde and sodium cyanoborohydride containing different isotopic labels
Figure RE-GDA0003308658500000021
In order to achieve the purpose, the invention adopts the technical scheme that:
1. preparation of internal standard amino acid: any one of 8 labeling combinations of labeled standard amino acids is used as an internal standard amino acid, and Dim36 labeled standard amino acids are selected in the kit.
2. Preparing a quality control product: labeling standard amino acid with labeling reagent different from the internal standard amino acid, and adding into the serum dry powder. The kit selectively uses Dim28 to mark standard amino acid, and adds internal standard amino acid.
3. Preparing a calibration product: dim28, Dim30H, Dim32H, Dim34H and Dim36 are selected to mark standard amino acids respectively and are mixed together in a certain concentration gradient and added into serum dry powder.
4. Clinical sample amino acid extraction: blood was taken intravenously, blood cells were removed by centrifugation, and the supernatant was collected. Adding acetonitrile or methanol into the supernatant to precipitate protein, centrifuging to obtain supernatant, blow drying, redissolving, and adding a labeling reagent to make free amino acid react with a chemical label. The labeling reagent cannot be identical to the internal standard amino acid.
5. And re-dissolving the quality control product and the calibrator. Adding acetonitrile or methanol, precipitating protein, and collecting supernatant.
6. Mass spectrometry analysis: and (4) drying the supernatants of the quality control product and the calibrator by using nitrogen, and redissolving by using a mobile phase A liquid of a chromatogram. And (3) respectively carrying out mass spectrum analysis on the clinical sample, the quality control product and the calibrator.
The invention has the following advantages:
1. the sample pretreatment is simple, and the sample pretreatment process of the method is the same as the conventional sample pretreatment process.
2. The marking method is simple, efficient, rapid, high in reaction effect and free of side reaction, and can finish marking of the sample within 5 minutes, and the marking efficiency is 100%.
3. The labeling method has low cost of labeling samples, and the labeling price of 100 mu mol of amino acid samples is much lower than the price of an iTRAQ reagent and the price of directly using stable isotope labeled amino acid.
Description of the drawings:
1. FIG. 1 is a schematic representation of the products and mass gain of amino acids labeled with formaldehyde and sodium cyanoborohydride of different isotopic composition.
Detailed Description
1. Preparation of internal standard amino acid: mixing 10 μ L4%13CD2O and 10. mu.L of 0.6mol/L NaCNBD3The labeling reagent of (2) was added to 100. mu.L of a 0.01M aqueous tyrosine solution, reacted at room temperature for 15min, followed by addition of 10. mu.L of 1M NH4HCO3The solution was reacted for 10min to remove excess labeling reagent. The solution after the above reaction was used as an internal standard stock solution, and the working solution was diluted 1000 times with 0.1% formic acid to be used as a working solution.
2. Preparing a quality control product: 10 μ L of 4% CH2O and 10. mu.L of 0.6mol/L NaCNBH3The labeling reagent of (2) was added to 100. mu.L of a 0.01M aqueous tyrosine solution, reacted at room temperature for 15min, followed by addition of 10. mu.L of 1M NH4HCO3The solution was reacted for 10min to remove excess labeling reagent, the working solution was diluted 500-fold with 0.1% formic acid, and 10. mu.L of the solution was added to the serum dry powder. Adding 10 μ L of internal standard working solution into the serum dry powder, dissolving the serum dry powder with 0.1mL of 0.1% formic acid, adding 400 μ L of acetonitrile to precipitate protein, centrifuging at 15000g, removing protein, collecting supernatant, blow-drying with nitrogen, and redissolving with 100 μ L of 0.1% formic acid for later use.
3. Preparing a calibration product: in the same manner, the tyrosine solutions were labeled with Dim28, Dim30H, Dim32H, Dim34H and Dim36 reagents, respectively. And the ratio of Dim 28: dim 30H: dim 32H: dim 34H: mixing Dim36 ═ 2:10:50:250:500, adding into 100 μ L serum solution, adding 400 μ L acetonitrile to precipitate protein, centrifuging at 15000g, removing protein, collecting supernatant, blowing with nitrogen, and redissolving with 100 μ L0.1% formic acid for use.
4. Clinical sample amino acid extraction: blood was collected intravenously, and the supernatant was collected by removing blood cells by centrifugation. Collecting 100 μ L supernatant, adding 400 μ L acetonitrile to precipitate protein15000g for 10min, collecting the supernatant, adding 10 μ L of 4% CH2O and 10. mu.L of 0.6mol/L NaCNBH3Reaction at room temperature for 15min, followed by addition of 10. mu.L of 1M NH4HCO3And (3) reacting the solution for 10min to remove redundant marking reagents, adding 10 mu L of internal standard working solution, desalting, drying, and redissolving by using 100 mu L of mobile phase A solution for later use.
5. Mass spectrometry analysis: and (3) respectively carrying out mass spectrum analysis on the clinical sample, the quality control product and the calibrator. Liquid chromatography conditions: ACQUITY UPLC I-Class with column oven (CH-A), sample size 1. mu.L, column temperature: 40 ℃; column ACQUITY UPLCR BEH C18,1.7 μm, 2.1X 100mm, flow rate 0.2mL/min, mobile phase A: water + 0.1% FA; mobile phase B: meoh + 0.1% FA +2mM ammonium form. Gradiometer:
Time ％B Curve
0 2 6
2 2 6
3.5 10 6
4 80 6
4.3 2 6
4.5 2 6
mass spectrum conditions: xevo TQD; acquisition mode MRM, parent ion 210> 164; 212> 166; 214> 168; 216> 170; 218> 172. Polarity ESI +; capillary voltage 0.5 kV; the ion source temperature is 150 ℃; desolventizing gas temperature: 350 ℃; cone voltage 16V, impact energy 16V.
Data processing: MassLynx software version 4.1 with TargetLynx application management software
Evaluation of the method:
1. external standard curve: the method utilizes the addition of the amino acid marked by five isotopes into the same sample and the mixing of the amino acid with different concentration gradients, thereby overcoming the defects of low linear correlation coefficient of a quantitative curve and large consumption of machine time caused by multiple sample injection. Tyrosine was labeled in this example with Dim28, Dim30H, Dim32H, Dim34H and Dim36, labeled as Dim 28: dim 30H: dim 32H: dim 34H: dim36 was mixed at 2:10:50:250:500 and quantified by fragment ion peak area.
2. Quantifying quality control products: the content of the amino acid marked by Dim28 in the quality control product is 2 times of that of the amino acid marked by the internal standard Dim 36. The actual measurement result is 1.98 times, which shows extremely high quantitative accuracy.
Use of the MAAQ method in clinical sample analysis
Serum of healthy persons selected from 10 physical examination samples was analyzed by the MAAQ method, and the tyrosine content was 57.4 + -7.9. mu. mol/L.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top/bottom", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "sleeved/connected," "connected," and the like are to be construed broadly, e.g., "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A multiple amino acid quantitative Method (MAAQ) is characterized in that formaldehyde, sodium cyanoborohydride and respective isotope forms thereof are organically combined to carry out chemical labeling on amino acid, so that the amino acid to be detected is provided with different mass labels, the peak intensity of a primary spectrum or a secondary spectrum of a mass spectrum is utilized to carry out quantitative analysis, the flow in an experiment is immobilized, and required reagents and consumables are assembled into a kit to facilitate popularization and use of the method.
2. The method of claim 1, wherein: the organic combination of formaldehyde and sodium cyanoborohydride and the isotope form thereof is carried out to label the peptide segments, namely four typesFormaldehyde (CH)2O，13CH2O，CD2O and13CD2o) and two sodium cyanoborohydrides (NaCNBH)3And NaCNBD3) The combination was performed to form 8 labels, resulting in different quality tags, as shown in the following table.
Figure FDA0003132929320000011
3. The method of claim 1, the amino acids being characterized by: the full-spectrum amino acids in organisms specifically include: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, gamma-aminobutyric acid, glycine, serine, taurine, tyrosine, alpha-aminocaproic acid, asparagine, aspartic acid, citrulline, glutamic acid, glutamine, ornithine, cysteine, cystathionine, homocysteine, alpha-aminon-butyric acid, alanine, anserine, beta-alanine, beta-aminoisobutyric acid, carnosine, ethanolamine, delta-hydroxylysine, hydroxylated proline, 1-methylhistidine, 3-methylhistidine, phosphoethanolamine, phosphoserine, proline, sarcosine, argininosuccinic acid, hydroxylated citrulline.
4. The method of claim 1, wherein: the mass spectrum acquisition method mainly comprises a triple quadrupole mass spectrum, a quadrupole-time-of-flight mass spectrum and a quadrupole-electrostatic orbitrap mass spectrum, and comprises Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM), selective ion detection (SIM) and full scan (full scan).
5. The kit of claim 1, wherein: the kit comprises reagents required by experiments and required consumables, wherein the reagents comprise a calibrator, a quality control product, serum dry powder, an isotope labeling reagent, four kinds of formaldehyde and two kinds of cyano sodium borohydride, 1mol/L ammonium bicarbonate solution (pH 7.8), 50mmol/L phosphate buffer solution, acetonitrile, methanol and a chromatographic mobile phase; the consumables included 1.7mL centrifuge tubes.
6. Kit according to claims 1 and 5, comprising the following processing steps:
1) preparation of internal standard amino acid: any one of 8 marker combinations is used as an internal standard amino acid, and the kit selects Dim36 marker standard amino acids;
2) preparing a quality control product: marking standard amino acid by using a marking reagent different from the internal standard amino acid, adding the standard amino acid into the serum dry powder, and selectively marking the standard amino acid by using Dim 28; adding internal standard amino acid;
3) preparing a calibration product: selecting Dim28, Dim30H, Dim32H, Dim34H and Dim36 as markers of standard amino acids, mixing the materials together at a ratio of 1:5:10:50:100, and adding the mixture into serum dry powder;
4) clinical sample amino acid extraction: taking blood from vein, centrifuging to remove blood cells, and collecting supernatant; adding acetonitrile or methanol into the supernatant to precipitate protein, taking the supernatant, drying, redissolving, and adding a labeling reagent to enable free amino acid to react to form a chemical label; and adding the internal standard substance of the step 1; the kit uses Dim28, Dim30L, Dim30H, Dim32L, Dim32H, Dim34L and Dim34H to mark clinical samples;
5) re-dissolving the quality control product and the calibrator; adding the quality control product and the calibrator obtained in the steps 2 and 3 into acetonitrile or methanol, precipitating protein, and taking supernatant;
6) mass spectrometry analysis: drying the supernatants of the quality control product and the calibrator obtained in the step 5 by using nitrogen, and redissolving by using mobile phase A liquid of a chromatogram; and (4) respectively carrying out mass spectrum analysis on the clinical sample, the quality control product and the calibrator obtained in the step (4) and the step (5).
7. The kit according to claim 6, the labeling method of step 4, having the following characteristics: when using the SIM or MRM mode of the serial quadrupole of claim 4, the labeling reagent is selected from Dim28, Dim30H, Dim32H, Dim 34H; when selecting PRM, SIM or full scan of electrostatic orbitrap mass spectrometry, the labeling reagents were selected from Dim28, Dim30L, Dim30H, Dim32L, Dim32H, Dim34L, Dim34H, while the mass spectral resolution was greater than 60000 when m/z is 200.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114740121A (en) * 2022-04-24 2022-07-12 杭州广科安德生物科技有限公司 Double-amino-acid stable isotope labeling method based on primary mass spectrometry and application

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114740121A (en) * 2022-04-24 2022-07-12 杭州广科安德生物科技有限公司 Double-amino-acid stable isotope labeling method based on primary mass spectrometry and application

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