CN115711936A - Detection method of amino acid and detection kit - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a detection method of amino acid and a detection kit for detecting amino acid. The amino acid detection method provided by the invention adopts a carboxyl-terminal long-chain polymer-biotin conjugate as a derivatization reagent to perform derivatization treatment on amino acid components in a sample, so that the amino acid components in the biological sample form amino acid derivatives; the amino acid derivatives were then detected using MALDI MS. The MALDI-MS method based on BPC derivatization is used for qualitative and quantitative detection of amino acid in a complex biological sample for the first time, and has the advantages of high specificity, accurate method, simple and convenient operation and high sensitivity.

Description

A kind of detection method of amino acid and detection kit

technical field

The invention relates to the technical field of biological detection, in particular to a method for detecting amino acids in biological samples, and also relates to a detection kit for detecting amino acids in biological samples.

Background technique

Amino acid is one of the three major nutrients of living organisms and the basic unit of enzymes and proteins. As core nutrients and participants in life metabolism, free amino acids in the body are of great significance to physiological functions and clinical diagnosis. The detection of free amino acids in biological samples is an important tool and work content in the fields of protein science, biochemistry, food science, and clinical medicine. For example, quantitative analysis of amino acids in plasma and dried blood film samples is indispensable for diagnosing inherited metabolic defects. Therefore, continuously improving the accuracy and work efficiency of amino acid analysis in biological samples and reducing detection costs are of great significance to related application fields, especially the clinical detection industry.

With the development of mass spectrometry, chromatographic separation and mass spectrometry have become the mainstream detection methods for amino acids in complex samples, including gas chromatography-mass spectrometry and liquid chromatography-electrospray-mass spectrometry. However, chromatography-mass spectrometry equipment is often complex and expensive, difficult to use and maintain, harsh to optimize conditions, and requires high professionalism of operators. More importantly, due to the existence of the chromatographic separation process, the analysis time is increased, the analysis throughput is reduced, and the cost of amino acid analysis is significantly increased. In the field of clinical mass spectrometry, matrix-assisted laser desorption ionization mass spectrometry (MALDIMS) is a high-throughput, automated mass spectrometry method with the advantages of high sensitivity, good stability, short experimental cycle, simple operation, and less consumption of samples and manpower. It has extremely high potential value for the detection of clinical indicators. However, there are three important problems in the detection of amino acids in complex samples (such as blood) by MALDI MS. First, during MALDI MS analysis, it is necessary to add ionized matrix molecules to the detection sample. Traditional matrix molecules will generate a series of high-sensitivity signals in the low molecular weight region, thereby significantly suppressing the signals of amino acid molecules that are also in this molecular weight region, reducing the The sensitivity and specificity of amino acid detection; second, because amino acid is a small molecular substance, there are fewer ionization sites in the structure, so its ionization efficiency is lower than that of macromolecular substances; In the molecular weight range, there are often other metabolic small molecules or salts. Since MALDI MS is generally not used in conjunction with chromatography, the presence of these interferences will inhibit the ionization and mass spectrometry signals of amino acids and reduce the analytical sensitivity.

In order to solve the above-mentioned problems in the detection of amino acids by MALDI MS, a feasible solution is to carry out specific chemical derivatization of amino acids. That is, by introducing a large molecular weight label structure that is easy to ionize on the amino acid, the ionization efficiency of the amino acid is improved, and the mass spectrometry signal of the amino acid is converted from the low molecular weight range to the higher molecular weight signal region, thereby avoiding the matrix in the low molecular weight range and other small molecules interfere with amino acid mass spectrometry signals, improving the sensitivity and specificity of amino acid detection. However, currently there are very few chemically derivatized candidate molecules that can simultaneously meet the above requirements (large molecular weight, easy ionization, mild, simple and rapid derivatization reaction) and are suitable for MALDI MS analysis.

Patent document CN1463291A discloses a method for detecting the relative amount of various biomolecules, which includes contacting the sample with an A-R affinity marker to obtain an affinity-labeled product, immobilizing the affinity-labeled product on a matrix through a capture reagent, and determining the labeled product amount. In this method, the A-R affinity tagged product is used as a capture reagent to capture the sample, and further includes the steps of capturing the affinity tagged product by the capture reagent and desorbing and ionizing the affinity tagged product. This method has many steps and is time-consuming, and requires specific capture reagents and multiple washing steps. In practical applications, it is not suitable for the analysis of small molecules such as amino acids.

Therefore, the detection accuracy and efficiency of amino acids in biological samples is still an important problem to be solved.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a method for detecting amino acids, which can detect amino acids in various biological samples, and the method is accurate, easy to operate and high in sensitivity.

The invention also provides a kit for amino acid detection.

In order to solve the above technical problems, in a first aspect, the present invention provides a method for detecting amino acids, comprising the steps of:

Using a carboxy-terminal long-chain polymer-biotin conjugate as a derivatization reagent, derivatizing the amino acid components in the biological sample, so that the amino acid components in the biological sample form amino acid derivatives;

Wherein, the carboxy-terminal long-chain polymer-biotin conjugate of the derivatization reagent used is the compound shown in formula I or formula II, or the terminal carboxyl group of the compound shown in formula I or formula II is further derivatized with succinimide or Compounds activated by succinimide analogues:

In formula I, m is an integer of 2 to 6;

In Formula II, n is an integer of 20-30.

The derivatization reagent used in the present invention is carboxy-terminal long-chain polymer-biotin conjugate (Biotin-Polymer-COOH, referred to as BPC), one end of the molecule is a carboxyl group, the other end is biotin, and the carboxyl group and biotin are polymerized material spacer. Preferably, the carboxyl terminus of the BPC is pre-activated with succinimide (NHS) or NHS analogues (such as "sulfonated NHS").

The molecular weight of the BPC can be changed by changing the molecular polymer chain length as required, that is, by selecting the value of m or n in the structural formula to select the "carboxyl-terminal long-chain polymer-biotin" with a specific molecular mass. "Conjugate" (BPC) is a derivatization reagent, which is used to derivatize amino acid components. Preferably, the molecular weight of the BPC ranges from 100 to 3000 Da.

When BPC is used as a derivatization reagent to derivatize the amino acid component, BPC can react with the amino group on the amino acid through the activated carboxyl group, and form a large molecular weight structural label by coupling on the amino acid. Therefore, when using MALDIMS for detection, the mass spectrum signal of amino acids can be increased from the low molecular weight range (m/z 50-250) to the required molecular weight range. Wherein, the required molecular weight range can be determined according to factors such as the matrix or sample type used in the detection, and the existing interfering substances. At the same time, since there are a large number of easily ionized structural sites in both the biotin structure and the polymer chain structure, the derivatized amino acids are very easy to form [M+H] ⁺ or [M+Na] ⁺ plasma in mass spectrometry The adduct increases the probability and efficiency of ionization, and can greatly improve the sensitivity of mass spectrometry to detect target signals.

As a preferred embodiment of the present invention, the present invention is mainly used for the detection of free amino acid components in biological samples, through the derivatization treatment of free amino acids, the free amino acid components in the biological samples are formed into amino acid derivatives, Then test it.

As a preferred embodiment of the present invention, the carboxy-terminal long-chain polymer-biotin conjugate is a compound represented by formula III or formula IV:

In formula III, m is an integer of 2 to 6;

In Formula IV, n is an integer of 20-30.

As a preferred embodiment of the present invention, the molecular weight of the carboxy-terminal long-chain polymer-biotin conjugate is in the range of 100-3000 Da.

As a preferred embodiment of the present invention, the compound represented by the formula III is selected from N-[6-(biotinamino)hexanoyl]-6-aminocaproic acid N-succinimide ester (Biotin-2AH- NHS, referred to as B2AN).

Wherein, the structural formula of N-[6-(biotinamino)caproyl]-6-aminocaproic acid N-succinimide ester (B2AN) is:

Using N-[6-(biotinylamino)caproyl]-6-aminocaproic acid N-succinimide ester as the derivatization reagent, the molecular weight of the amino acid can be increased by about 452 by derivatizing with the molecule, so that the mass spectrum of the amino acid The signal mainly appears in the range of m/z500-700, and will not be interfered by the signal of MALDI matrix molecules (the signal interference of small molecule matrix mainly appears in the range of m/z100-400). At the same time, because the B2AN structure has a large number of polar groups and sites that are easy to ionize, compared with underivatized amino acid molecules, the derivatized molecular structure is easier to ionize, which improves the ionization efficiency and mass spectrometry detection sensitivity. Therefore, amino acids in complex samples can be directly detected without pre-purification and separation.

As a preferred embodiment of the present invention, the compound represented by formula IV is selected from NHS-activated carboxyl polyethylene glycol-24-biotin conjugate (Biotin-PEG24-NHS, BP24N for short).

Wherein, the structural formula of NHS-activated carboxypolyethylene glycol-biotin conjugate (BP24N) is:

Using NHS-activated carboxypoly-24ethylene glycol-biotin conjugate as a derivatization reagent, amino acids can be derivatized with this molecule to increase the molecular weight to about 1375, so that the mass spectrometry signals of amino acids mainly appear in the m/z 1400-1700 interval , not only will not be interfered by MALDI matrix molecular signals (signal interference of small molecule matrix mainly appears in the m/z 100-400 range), but also not easily affected by other common high-abundance small molecule substances (such as phospholipids) in the sample matrix interference, the detection accuracy is significantly improved. Similarly, due to the large number of polar groups and easy-to-ionize sites in the BP24N structure, the derivatized molecular structure is easier to ionize than underivatized amino acid molecules, which improves ionization efficiency and mass spectrometry detection sensitivity.

As a preferred embodiment of the present invention, during the derivatization treatment, the derivatization reagent is prepared in the form of a solution for use, and the concentration of the derivatization reagent in the prepared solution is 0.1 mM˜100 mM.

Preferably, the reaction temperature during the derivatization treatment is 20°C to 40°C. The reaction time is preferably 5 min to 120 min.

As a preferred embodiment of the present invention, the detection method further includes the step of: using MALDI MS to detect the amino acid derivatives.

Furthermore, the detection method of the present invention further includes the step of: performing purification treatment on the biological sample before the derivatization treatment, preferably a purification treatment for removing macromolecular proteins. After purification, the interfering substances can be reduced, and the accuracy of detection can be further improved. Of course, biological samples can also be directly derivatized without purification to remove macromolecular proteins.

Preferably, the methods for removing or reducing high-abundance proteins, salts and other substances in the sample that interfere with MALDI MS detection include organic solvent precipitation, strong acid or ultra-speed filtration and centrifugation.

Among them, the organic solvent precipitation method can use organic solvents such as methanol or acetonitrile to remove proteins in plasma samples.

In a specific embodiment, the method used to remove macromolecular proteins is methanol precipitation protein method.

A specific operation step includes: taking a biological sample, adding methanol to sufficiently vortex and oscillate to precipitate protein, and then centrifuging to obtain the supernatant.

In a specific embodiment, the method used to remove macromolecular proteins is ultrafiltration centrifugation.

A specific operation step includes: taking a biological sample, adding MES buffer solution and mixing evenly, adding the mixed solution into an ultrafiltration centrifuge tube for centrifugal filtration (10000g/20min), removing high molecular weight proteins in the sample, and placing the ultrafiltration tube The plasma ultrafiltrate in the lower layer was collected.

Furthermore, the detection method of the present invention also includes the step of: after the derivatization treatment, purifying or desalting the biological sample. Of course, the derivatized samples can also be directly detected by MALDI MS without any treatment.

As a preferred embodiment of the present invention, the detection adopts an internal standard method for quantitative detection, and the internal standard used is an isotope-labeled amino acid.

In some embodiments, the internal standard employed is an isotopically labeled amino acid. The labeled isotope of the internal standard can be selected from isotopes such as ¹⁵ N, deuterium (D), ¹³ C and the like.

Typically, the heavy isotopes contained in isotopically labeled amino acids contain more than one heavy isotope. Incorporation of a higher number of heavy isotopes may be preferred as it provides greater mass shift. Such heavy isotopically labeled compounds are well known in the art and are available from a number of suppliers.

During the detection process, the detection process is carried out according to the internal standard method process.

In some embodiments, an isotopically labeled amino acid, ie, an internal standard, is added to the biological sample or standard solution prior to the derivatization reaction. On the one hand, the internal standard helps to quantitatively analyze the amino acids in the sample, and on the other hand, it can calibrate or correct the inaccurate results caused by the loss of the target substance or the matrix effect of the interfering substance during the experiment.

In some embodiments, according to the MS signal-to-noise ratio of amino acid standards and internal standards and the concentration of added amino acids, a standard curve is drawn to calculate the concentration of the amino acid in the biological sample.

In some embodiments, the amino acid derivatives are analyzed by MALDI-MS during detection, for example, a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer can be used for identification in reflectance mode. MALDI mass spectra were accumulated and averaged over 400 shots in positive ion mode at a laser intensity of 100-120. Shimadzu Biotech Launchpad software (version 2.9) is used for spectrum acquisition and processing, and other commercially available MALDI-MS mass spectrometers can also be used, which is not limited in this disclosure.

In the second aspect, the present invention also provides a kit for amino acid detection used in the detection method, comprising the carboxy-terminal long-chain polymer-biotin conjugate derivatization reagent.

As a preferred embodiment of the present invention, the kit further includes at least one of components i) to iii):

i) Reagents and consumables required for amino acid extraction and/or purification from biological samples;

ii) Reagents and consumables used for mass spectrometry detection;

iii) said amino acids labeled with isotopes and amino acid standards for quantitative detection.

In the third aspect, the present invention also provides the application of the detection method and the kit in detecting the content of amino acid components in amino acid-containing biological samples, especially the application in detecting the content of free amino acid components in biological samples .

As a preferred embodiment of the present invention, the amino acid-containing biological sample is any one of amino acid solution, dried blood film, blood, serum, plasma, urine, saliva, and interstitial fluid. Preferably, the biological sample is a blood sample. The blood samples include dried blood slices, blood, serum or plasma.

In some embodiments, the biological sample is a dried blood film sample. Dried blood films are samples of whole blood types including blood cells.

The amino acid that can be detected in the present invention is any amino acid, such as glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, Tyrosine, Cysteine, Phenylalanine, Asparagine, Glutamine, Threonine, Aspartic Acid, Glutamic Acid, Lysine, Arginine, Histidine, Selenium cysteine and pyrrolysine.

The detection method of amino acid provided by the present invention has the following beneficial effects:

1) It can completely solve the main problems in the amino acid detection by MALDI MS. Most of the existing amino acid derivatization methods are developed for chromatography, spectroscopy, GC-MS, LC-MS and other techniques, which cannot specifically solve the problems in the detection of amino acids by MALDI MS. The amino acid derivatization method provided by the present invention can specifically solve the problems of matrix and background interference and low amino acid ionization efficiency in MALDI MS detection, and complex samples can be realized only by using the most classic MALDI MS process and a general-purpose MALDI matrix. The amino acid in the method is directly detected, without additional modification of the matrix, and without the use of capture reagents to capture the derivatization reagents and/or samples, with short reaction time and few operating steps, avoiding sample loss or dilution caused by multiple washings, and has the advantages of Extremely high utility potential.

2) The sample preparation is simple and clear, the method is simple, the running time of the instrument is short, and the degree of automation is high. Existing GC-MS and LC-MS methods require complex method establishment, optimization and validation processes, strong chromatography and mass spectrometry background knowledge and professional operators, but many clinical institutions that need to determine amino acids in biological samples do not have such a condition. The method provided by the invention combines BPC derivatization with MALDI MS, improves the ionization efficiency, and improves the detection sensitivity of MALDI-MS. And without any chromatographic separation or enrichment process, the operation time is short, and it is easy to realize high-throughput detection of biological samples.

3) BPC with a suitable molecular weight can be selected as a derivatization reagent according to needs. Since the background matrix that interferes with amino acid detection in different samples is different, the molecular weight of amino acid derivatives can be changed by adjusting the number of polymer spacers in BPC, so as to adjust the target signal to a specific molecular weight region with less background interference. Therefore, such derivatization reagents can be applied to different sample types due to their structural flexibility.

The MALDI-MS method based on BPC derivatization of the present invention is used for the qualitative and quantitative detection of amino acids in complex biological samples for the first time, and has high specificity, accurate method, simple operation and high sensitivity. Experimental results show that the method of the present invention can be used for clinical determination of amino acids in complex biological samples.

Description of drawings

Fig. 1 is the mass spectrometry signal spectrogram that detects with MALDI MS after the lysine standard substance is derivatized by B2AN in the embodiment 2 of the present invention;

Fig. 2 is the mass spectrum signal spectrum of the arginine standard substance detected by MALDI MS after being derivatized by B2AN in Example 2 of the present invention;

Fig. 3 is the detection mass spectrogram of plasma sample in the embodiment 3 of the present invention;

Fig. 4 is the detection mass spectrogram of dried blood sample in embodiment 4 of the present invention;

Fig. 5 is the detection mass spectrogram of dried blood sample in embodiment 5 of the present invention;

Fig. 6 is the quantitative standard curve diagram of different amino acids in Example 6 of the present invention;

Fig. 7 is the detection mass spectrogram of plasma samples in Example 7 of the present invention derivatized with B2AN and derivatized without derivatization;

Fig. 8 is a detection mass spectrum of plasma samples derived from BPC molecules with three different molecular weights in Example 8 of the present invention.

Detailed ways

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment.

Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the invention are disclosed in or are apparent from the following detailed description. It is to be understood by those of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended to limit the broader aspects of the invention.

In the following examples of the present invention, N-[6-(biotinamino)caproyl]-6-aminocaproic acid N-succinimide ester (B2AN) or NHS-activated carboxyl polyethylene glycol-24-bio The prime conjugate (BP24N) is a derivatization reagent, and the content of amino acid components in several biological samples has been detected. The detection method adopts the internal standard method for quantitative detection, and the internal standard substance used is isotope-labeled amino acid.

First, the amino acid in the biological sample is derivatized to obtain amino acid derivatives; when the amino acid in the biological sample is derivatized, the concentration of the derivatization reagent used is 0.1mM-100mM; the reaction temperature of the derivatization treatment is 20℃～40℃;

The amino acid derivatives were then detected using MALDI MS.

Preferably, before the derivatization treatment, the biological sample is subjected to purification treatment, for example, the purification treatment includes purification treatment for removing macromolecular proteins.

Preferably, after the derivatization treatment, the derivatized biological sample is purified or desalted.

The technical solution of the present invention will be described in detail below through specific examples.

Example 1

This embodiment provides a method for detecting free amino acids in plasma samples, and the specific steps are as follows:

1) Take 30 μL of plasma sample, add 200 μL of methanol to fully vortex and oscillate to precipitate protein, centrifuge to absorb the supernatant, put it into a new ep tube, and vacuum dry. Add 20 μL of pH 8.0 MES buffer solution and mix well to obtain the processed plasma sample.

B2AN was dissolved in acetonitrile/H ₂ O (1:1, v/v) mixed solution to prepare a B2AN solution, wherein the concentration of B2AN was 20 mM.

2) Take 18 μL of the above-mentioned processed plasma sample and blank MES buffer solution, add 2 μL of B2AN solution respectively, and react at room temperature for 30 minutes.

3) Take 5 μL of the reaction solution obtained after the reaction in step 2) respectively, and then perform the following treatments on the reaction solution respectively:

Dilute 5 times with acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v) mixed solution;

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Aspirate 1.5 μL of the test sample, and spot the test sample solution on the surface of the stainless steel target plate. Amino acid derivatives were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode for identification.

MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode with a laser intensity of 100-120. Use Shimadzu Biotech Launchpad software (version 2.9) for spectrum acquisition and processing.

Example 2

This example investigates the feasibility of N-[6-(biotinamino)caproyl]-6-aminocaproic acid N-succinimide ester (abbreviated as B2AN) derived amino acid standard. Taking arginine and lysine as examples, the steps are as follows:

1) Prepare arginine and lysine standards with MES buffer solution at pH 8.0 to prepare 100 μM solutions. B2AN was dissolved in acetonitrile/H ₂ O (volume ratio 1:1) mixed solution, and the concentration of B2AN was prepared at 20 mM.

2) Take 18 μL of arginine and lysine standard solution and blank MES buffer solution respectively, and add 2 μL of B2AN solution respectively. Thereafter, the reaction was carried out at room temperature for 30 minutes. The derivatization reaction equations of lysine, arginine and B2AN are as follows:

The molecular weights of lysine and arginine were 146.11 and 174.11, respectively. After derivatization with B2AN, the molecular weights of the detected molecules were 598.35 and 626.36, respectively. Therefore, the mass spectrometric signals of both molecules will appear in the range above m/z 500, where the signal of the detected molecule will not be interfered by the background signal of the MALDI matrix (commonly used MALDI matrices such as dihydroxybenzoic acid background signal interference usually occurs in the range of m/z 100-400).

3) Take 5 μL of the reaction solution obtained from the reaction in step 2) and dilute it 5 times with a mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate, analyze the amino acid derivatives by MALDI-MS, and use the SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer to identify them in reflection mode. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Shimadzu Biotech Launchpad software (version 2.9) was used for spectrum collection and processing, and the analysis results are shown in Figure 1 and Figure 2. Among them, Fig. 1 is the mass spectrometry signal detected by MALDI MS and the control spectrogram of the blank sample after the lysine standard is derivatized by B2AN, and Fig. 2 is the mass spectrometry signal detected by MALDI MS after the arginine standard is derivatized by B2AN and Comparison spectrum with blank sample.

Figures 1 and 2 show that: MALDI MS can detect the signals of B2AN derivatives of arginine and lysine with high sensitivity. On the contrary, the blank sample has no obvious interference peaks in this signal area. This result shows that it is feasible to derivatize amino acids with B2AN and use the signal of B2AN derivatives to detect and quantify amino acids; through B2AN derivatization, the molecular weight of amino acids can be raised to the range above m/z 500, within which There is basically no interference from the matrix signal, which can greatly improve the sensitivity and ionization efficiency of amino acid detection, and realize the highly sensitive detection of MALDI MS. There is no interference from the matrix background signal in the detected area, and the detection process does not require complicated chromatographic separation and purification. .

Example 3

This example provides a method for detecting free amino acids in plasma samples. B2AN is used for derivatization and MALDI MS for detection. The specific steps are as follows:

1) Take 60 μL of plasma sample, add an equal volume of MES buffer solution with pH 8.0 and mix well. The mixed solution was added to a 3kd ultrafiltration centrifuge tube for centrifugal filtration (10000g/20min) to remove high molecular weight proteins in the sample. Collect the plasma ultrafiltrate in the lower layer of the ultrafiltration tube into a new EP tube.

B2AN was dissolved in acetonitrile/H ₂ O (volume ratio 1:1) mixed solution, and the concentration of B2AN was prepared at 20 mM.

2) Take 18 μL of the above plasma ultrafiltrate and blank MES buffer solution, add 2 μL of B2AN solution respectively, and react at room temperature for 30 minutes.

3) Take 5 μL of the reaction solution obtained in step 2) and dilute it 5 times with a mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate, analyze the amino acid derivatives by MALDI-MS, and use the SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer to identify them in reflection mode. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Using Shimadzu Biotech Launchpad software (version 2.9) for spectrum collection and processing, the results are shown in Figure 3. Figure 3 (a) shows the mass spectrum of the plasma sample that has been derivatized with B2AN and free amino acids detected by MALDI MS after ultrafiltration and centrifugation to remove high-abundance proteins. Figure 3 (b) is the mass spectrum of the blank control sample picture.

It can be seen from Figure 3 that most of the different kinds of amino acid molecules in plasma are detected as [M+H] ⁺ or [M+Na] ⁺ ions and are marked in the figure. In contrast, no corresponding signal appeared in the blank sample. In this study, free amino acids in plasma can be directly and highly sensitively detected by MALDI MS through the derivatization reaction of BPC molecules (B2AN in this figure), without any complicated chromatographic separation and purification steps.

By derivatizing amino acids with B2AN, MALDI MS can directly detect the signals of different kinds of amino acids in plasma samples without any chromatographic separation or purification process, and the whole process is simple and fast. This result shows that it is feasible to derivatize amino acids with B2AN and use the signals of B2AN derivatives to detect amino acid components in complex sample matrices. Through B2AN derivatization, the molecular weight of amino acids can be increased to a range above m/z 500, and there is basically no interference from matrix signals in this range, which can greatly improve the sensitivity and ionization efficiency of amino acid detection.

Example 4

This embodiment provides a method for detecting amino acids in dried blood samples, and the specific steps are as follows:

1) A blood sample with a diameter of 5 mm is separated from the dried blood sheet by punching, and placed in an EP tube. Add 100 μL of methanol to the tube, vortex for 10 minutes, take out the methanol clear solution and place it in a new EP tube, and vacuum the solvent to dry up. At the same time prepare corresponding blank paper samples.

B2AN was dissolved in acetonitrile/H ₂ O (volume ratio 1:1) mixed solution, and the concentration of B2AN was prepared at 20 mM.

2) Add 18 μL of MES buffer solution with a pH of 8.0 to the sample tube of the dried blood slice extract, vortex and oscillate sufficiently, then add 2 μL of B2AN solution, and mix well. React at room temperature for 30 minutes.

3) Take 5 μL of the reaction mixture obtained in step 2), and dilute it 5 times with the mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution to spot the surface of the stainless steel target plate. Amino acid derivatives were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode for identification. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Use Shimadzu Biotech Launchpad software (version 2.9) for spectrum acquisition and processing. The result is shown in Figure 4. Figure 4 (a) shows the mass spectrum of the dried blood sample after methanol extraction, derivatization with B2AN, and detection of free amino acids in it by MALDI MS, and Figure 4 (b) is the mass spectrum of the blank control sample.

It can be seen from Figure 4 that most of the different types of amino acid molecules in dried blood slices are detected as [M+H] ⁺ or [M+Na] ⁺ ions and are marked in the figure. In contrast, no corresponding signal appeared in the blank sample. In this study, free amino acids in dried blood slices can be directly and highly sensitively detected by MALDI MS through the derivatization reaction of BPC molecules (B2AN in this figure), without any complicated chromatographic separation and purification steps.

By derivatizing amino acids with B2AN, MALDI MS can directly detect different types of amino acid signals in dried blood samples without any chromatographic separation or purification process, and the whole process is simple and fast. This result shows that it is feasible to use B2AN to derive amino acids and use the signals of B2AN derivatives to detect amino acid components in different types of complex sample matrices, and the signal has a high signal-to-noise ratio and good detection sensitivity.

Example 5

In order to investigate the derivatization and detection effects of carboxypolyethylene glycol-biotin conjugates (BPC) with different molecular weights on amino acids, this example specifically tested the NHS-activated carboxypolyethylene glycol-biotin conjugates (BP24N ) to derive and detect the feasibility of amino acids in dried blood film samples, the specific steps are:

1) Separate the blood sample with a diameter of 5 mm from the dried blood film by punching, place it in an EP tube, add 100 μL of methanol to the tube, vortex for 10 minutes, take out the methanol clear solution and place it in a new EP tube. In the tube, the solvent was dried in a vacuum. At the same time, prepare corresponding blank paper samples.

Dissolve BP24N in pure water, and the prepared concentration of BP24N is 20mM.

2) Add 18 μL of MES buffer solution with a pH of 8.0 to the sample tube of the dried blood slice extract, vortex and oscillate sufficiently, then add 2 μL of BP24N solution, and mix well. React at room temperature for 30 minutes.

3) Take 5 μL of the reaction solution obtained in step 2), and dilute it 5 times with a mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate. Amino acid derivatives were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode for identification. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Using Shimadzu Biotech Launchpad software (version 2.9) for spectrum collection and processing, the results are shown in Figure 5. Figure 5(a) shows the mass spectrum of the dried blood sample sample extracted with methanol, derivatized with NHS-activated carboxypolyethylene glycol-biotin conjugate (BP24N) and detected by MALDI MS, (b) in Fig. 5 is the result of the blank control sample.

Most of the different kinds of amino acid molecules in dried blood slices were detected as [M+K] ⁺ or [M+Na] ⁺ ions and marked in the figure. In contrast, no corresponding signal appeared in the blank sample. In this study, free amino acids in dried blood slices can be directly and highly sensitively detected by MALDI MS through the derivatization reaction of BPC molecules (BP24N in this figure), without any complicated chromatographic separation and purification steps.

Through BP24N derivatization, MALDI MS can directly detect different kinds of amino acid signals in dried blood samples without any chromatographic separation or purification process, and the whole process is simple and fast. This result also shows that it is feasible to derivatize and detect amino acid components in complex sample matrices with carboxypolyethylene glycol-biotin conjugates (BPC) of different molecular weights. According to the type of sample matrix, the molecular weight of interfering substances, etc., BPC with an appropriate molecular weight can be selected to derivatize amino acids, so that the signal of amino acids can be converted to a molecular weight range with less interference to obtain the best detection sensitivity and specificity.

Example 6

This example investigates the feasibility of B2AN derivatization combined with MALDI MS detection for the quantitative analysis of free amino acids in samples and the determination of the standard curve, the steps are as follows:

1) Prepare a 25 μM lysine isotope internal standard solution with MES buffer solution at pH 8.0, and the lysine isotope internal standard contains six ¹³ C isotopes. Use this internal standard buffer solution to dilute and prepare amino acid mixed standard solutions with different concentrations. The amino acid mixed standard solution contains different kinds of amino acid standards, including glycine, alanine, threonine, proline, lysine, amino acid, phenylalanine, arginine. The concentration of each amino acid standard is slightly different, and the overall concentration is in the range of 0.1-80 μM.

B2AN was dissolved in acetonitrile/H ₂ O (1:1) mixed solution, and the prepared concentration of B2AN was 20 mM.

2) Take 18 μL of the above amino acid mixed standard solution and add 2 μL of B2AN solution respectively, and react at room temperature for 30 minutes.

3) Take 5 μL of the reaction mixture and dilute it 5 times with the mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate, analyze the amino acid derivatives by MALDI-MS, and use the SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer to identify them in reflection mode. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Use Shimadzu Biotech Launchpad software (version 2.9) for spectrum acquisition and processing. All spectrograms were normalized using the lysine isotope internal standard. The amino acid standard concentration was taken as the abscissa, and the mass spectrum response value of the amino acid derivative signal was used as the ordinate to draw the quantitative standard curve of amino acids. The results are shown in Figure 6. Show. Figure 6 shows the standard curve for the quantitative analysis of amino acids using the method of the present invention, a mixed standard solution of eight amino acids was tested, and the isotope standard of lysine was used as the internal standard. The solution contains different kinds of amino acid standards, including glycine, alanine, threonine, proline, lysine, histidine, phenylalanine, arginine, the concentration of each amino acid standard is slightly There are differences, and the overall concentration range is in the range of 0.1-80 μM. The results show that by combining B2AN and MALDI MS detection, an ideal amino acid quantitative standard curve can be obtained. The ^R2 of all amino acid standard curves are above 0.99, and the response values of each concentration have good reproducibility, and the CV% is 20%. Below, the requirements for amino acid quantitative analysis can be met.

Example 7

This example compares the difference in the effect of using MALDI MS to detect plasma amino acids under the two treatment conditions of BPC (with B2AN as the derivatization reagent) and without any derivatization. The specific operation steps are:

1) Take two plasma samples, each with 30 μL plasma. Add 200 μL methanol to the two samples respectively to precipitate high molecular weight proteins in the samples. The mixture was shaken and mixed for 2 minutes, then centrifuged at 10000g for 20 minutes, and the supernatant in the sample tube was transferred into two new EP tubes. The solvent in both tubes was then vacuum-dried.

B2AN was dissolved in acetonitrile/H ₂ O (volume ratio 1:1) mixed solution, and the concentration of B2AN was prepared at 20 mM.

2) Add 18 μL of MES buffer solution with a pH of 8.0 to the above two sample tubes, and after fully vortexed, add 2 μL of B2AN solution to one of the sample tubes and mix well. React at room temperature for 30 minutes. Add 2 μL of blank aqueous solution to another sample tube.

3) Take 5 μL of each of the two reaction mixtures obtained in step 2), and dilute 5 times with the mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate, analyze the amino acid derivatives by MALDI-MS, and use the SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer to identify them in reflection mode. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Using Shimadzu Biotech Launchpad software (version 2.9) for spectrum collection and processing, the results are shown in Figure 7. (a) in Figure 7 shows the mass spectrum of free amino acids detected by MALDI MS after B2AN derivatization, and (b) in Figure 7 is the mass spectrum of the region corresponding to the molecular weight of amino acids detected by MALDI MS without any derivatization (theoretical The underivatized amino acid signal is in the m/z 50-300 range).

It can be seen from Figure 7 that the highly sensitive plasma free amino acid signal can be stably obtained by BPC derivatization and MALDI MS detection; on the contrary, in the case of no derivatization, no credible amino acid signal can be detected by MALDI MS in the same sample, The main signal in mass spectrometry comes from background components such as matrix molecules such as DHB.

Example 8

This embodiment compares the plasma samples derived from BPC molecules with different spacer chain lengths and different molecular weights, and then uses MALDI MS to detect the effect of plasma amino acids. The three BPC molecules are: N-[6-(biotinamino )hexanoyl]-6-aminocaproic acid N-succinimide ester (abbreviated as B2AN, molecular weight 567.7Da), N-succinimidyl 6-biotinaminocaproic acid (abbreviated as B1AN, molecular weight 454.5Da), and D -Biotin N-hydroxysuccinimide ester (abbreviated as B0AN, molecular weight 341.4Da). The specific operation steps are:

1) Take three plasma samples, each with 30 μL plasma. Add 200 μL methanol to the two samples respectively to precipitate high molecular weight proteins in the samples. The mixture was shaken and mixed for 2 minutes, then centrifuged at 10000g for 20 minutes, and the supernatant in the sample tube was transferred into three new EP tubes respectively. The solvent in the three tubes was then vacuum-dried.

B2AN, B1AN, and B0AN were respectively dissolved in a mixed solution of acetonitrile/H ₂ O (volume ratio 1:1) at a concentration of 20 mM.

2) Add 18 μL of MES buffer solution with a pH of 8.0 to the above three sample tubes, and after fully vortexed, add 2 μL of B2AN, B1AN, and B0AN solutions to the three sample tubes, and mix well. React at room temperature for 30 minutes.

3) Take 5 μL each of the three reaction mixtures obtained in step 2), and dilute 5 times with the mixed solution of acetonitrile/water/trifluoroacetic acid (1:1:0.002, v/v/v);

Mix 1.5 μL of the diluent with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare a test sample, wherein the 2,5-dihydroxybenzoic acid solution is 2,5-dihydroxybenzoic acid in acetonitrile/water/ Trifluoroacetic acid (1:1:0.002, v/v/v) solution, the concentration is 10mg/mL;

Pipette 1.5 μL of the test sample solution onto the surface of the stainless steel target plate, analyze the amino acid derivatives by MALDI-MS, and use the SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer to identify them in reflection mode. MALDI mass spectra were obtained by accumulating and averaging 400 shots in positive ion mode at a laser intensity of 100-120. Using Shimadzu Biotech Launchpad software (version 2.9) for spectrum acquisition and processing, the results are shown in Figure 8. (a), (b) and (c) in Figure 8 show the mass spectra of free amino acids detected by MALDI MS after derivatization of B2AN, B1AN and BOAN, respectively.

It can be seen from Figure 8 that a certain amount of plasma free amino acid signals can be collected by BPC derivatization with different molecular weights; however, we also found that as the molecular weight of BPC decreases, the detectable amino acid signals decrease significantly, especially B0AN with a molecular weight of 341 , only two or three amino acid signals with high signal-to-noise ratio can be detected after derivatization. This is due to the fact that there are many background interference signals in the range of m/z 300-500 in the plasma sample. These signals come from the lipids, salts and other high-abundance substances in the plasma on the one hand, and from the MALDI mass spectrometry matrix on the other hand. background peak. Since BOAN derivatization cannot elevate the amino acid signal to a higher molecular weight range, the detection effect is not ideal under the interference of these strong background signals. This result shows that in order to obtain ideal detection results, the molecular weight of the amino acid derivatization reagent should not be too small, and the derivatization reaction must at least be able to increase the detection signal of the amino acid to a range above m/z 500 (such as B2AN), in order to achieve a more reliable High sensitivity analysis. The various amino acid derivatization reagents that have been developed in the past have molecular weights below 300 Da, and are not suitable for MALDI MS detection of complex samples.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (12)

1. A detection method for amino acids, comprising the steps of: Using a carboxy-terminal long-chain polymer-biotin conjugate as a derivatization reagent, derivatizing the amino acid components in the biological sample, so that the amino acid components in the biological sample form amino acid derivatives; The carboxy-terminal long-chain polymer-biotin conjugate is the compound shown in formula I or formula II, or the terminal carboxyl group of the compound shown in formula I or formula II is further replaced by succinimide or succinimide Phytoactivated compounds:

In formula I, m is an integer of 2 to 6; In Formula II, n is an integer of 20-30. 2. The detection method according to claim 1, characterized in that, the carboxy-terminal long-chain polymer-biotin conjugate is a compound shown in formula III or formula IV:

3. The detection method according to claim 1 or 2, characterized in that the molecular weight range of the carboxy-terminal long-chain polymer-biotin conjugate is 100-3000 Da. 4. detection method according to claim 3, is characterized in that, the compound shown in described formula III is selected from N-[6-(biotinamino) hexanoyl]-6-aminocaproic acid N-succinimide ester; and/or, the compound represented by the formula IV is selected from NHS-activated carboxypoly-24ethylene glycol-biotin conjugates. 5. The detection method according to any one of claims 1 to 4, characterized in that, when performing the derivatization treatment, the concentration of the derivatization reagent used is 0.1 mM to 100 mM; preferably, the derivatization The reaction temperature of the chemical treatment is 20°C to 40°C. 6. The detection method according to any one of claims 1-5, characterized in that, the detection method further comprises the step of: using MALDI MS to detect the amino acid derivatives. 7. detection method according to claim 1 or 6, is characterized in that, described detection method also comprises the step: Before the derivatization treatment, the biological sample is purified, preferably to remove macromolecular proteins; More preferably, after the derivatization treatment, the biological sample is purified or desalted. 8. The detection method according to claim 6 or 7, characterized in that the detection adopts an internal standard method for quantitative detection, and the internal standard used is an isotope-labeled amino acid. 9. A kit for amino acid detection used in the detection method according to any one of claims 1 to 8, characterized in that it comprises the carboxy-terminal long-chain polymer-biotin conjugate. 10. The kit according to claim 9, characterized in that, the kit further comprises at least one of i) to iii) components: i) Reagents and consumables required for amino acid extraction and/or purification from biological samples; ii) Reagents and consumables used for mass spectrometry detection; iii) Isotope-labeled amino acids and amino acid standards for quantitative detection. 11. Application of the detection method according to any one of claims 1 to 8 and the kit according to claim 9 or 10 in detecting the content of amino acid components in biological samples. 12. The application according to claim 11, wherein the biological sample is any one of amino acid solution, dried blood film, blood, serum, plasma, urine, saliva, and interstitial fluid.

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