Method for detecting amino acid and kit for detection

Abstract

The application belongs to the technical field of biological detection, and specifically discloses a kind of amino acid detection method, and simultaneously discloses a kind of detection reagent kit for detecting amino acid.The amino acid detection method provided by the application uses carboxyl terminal long-chain polymer-biotin conjugate as a derivatization reagent to perform derivatization treatment on amino acid components in a sample, so that amino acid components in the biological sample form amino acid derivatives; then MALDI MS is used to detect the amino acid derivatives.The MALDI-MS method based on BPC derivatization is used for the first time for qualitative and quantitative detection of amino acids in complex biological samples, and has high specificity, accurate method, simple operation, and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for detecting amino acids in biological samples, as well as a detection kit for detecting amino acids in biological samples. Background Technology

[0002] Amino acids are one of the three major nutrients for living organisms and are the basic building blocks of enzymes and proteins. As core nutrients and participants in metabolism, free amino acids in the body are of great significance for physiological functions and clinical diagnosis. The detection of free amino acids in biological samples is an important tool and task in fields such as protein science, biochemistry, food science, and clinical medicine. For example, quantitative analysis of amino acids in plasma and dried blood smears is indispensable for diagnosing inherited metabolic disorders. Therefore, continuously improving the accuracy and efficiency of amino acid analysis in biological samples and reducing testing costs is of great significance for related application fields, especially the clinical testing industry.

[0003] With the development of mass spectrometry technology, chromatographic separation coupled with mass spectrometry has become the mainstream method for detecting amino acids in complex samples, including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). However, GC-MS equipment is often complex and expensive, difficult to use and maintain, requires stringent optimization conditions, and demands a high level of expertise from operators. More importantly, the chromatographic separation process increases analysis time and reduces throughput, significantly increasing the cost of amino acid analysis. In the field of clinical mass spectrometry, matrix-assisted laser desorption / ionization mass spectrometry (MALDIMS) is a high-throughput, automated mass spectrometry method with advantages such as high sensitivity, good stability, short experimental cycle, simple operation, and low sample and manpower consumption, making it highly valuable for the detection of clinical indicators. However, there are three important problems with using MALDIMS for the detection of amino acids in complex samples (such as blood). First, MALDI MS analysis requires the addition of matrix molecules to the sample for ionization. Traditional matrix molecules generate a series of highly sensitive signals in the low molecular weight region, which significantly suppresses the signals of amino acid molecules in the same molecular weight region, reducing the sensitivity and specificity of amino acid detection. Second, because amino acids are small molecules, they have fewer ionization sites in their structure, resulting in lower ionization efficiency compared to large molecules. Third, complex samples often contain other small metabolic molecules or salts within the molecular weight range of amino acids. Since MALDI MS is generally not coupled with chromatography, the presence of these interfering substances will inhibit the ionization of amino acids and the mass spectrometry signal, reducing analytical sensitivity.

[0004] To address the aforementioned issues in MALDI MS detection of amino acids, a feasible approach is to specifically chemically derivatize the amino acids. This involves introducing a readily ionizable, high-molecular-weight tag structure onto the amino acid to enhance ionization efficiency and shift the mass spectrometric signal from the low molecular weight range to a higher molecular weight signal region. This avoids interference from low-molecular-weight matrix molecules and other small molecules, thereby improving the sensitivity and specificity of amino acid detection. However, currently, very few chemically derived candidate molecules simultaneously meet the above requirements (high molecular weight, readily ionizable, mild, simple, and rapid derivatization reaction) and are suitable for MALDI MS analysis.

[0005] Patent document CN1463291A discloses a method for detecting the relative amounts of multiple biomolecules, including contacting a sample with an AR affinity label to obtain an affinity-labeled product, immobilizing the affinity-labeled product on a matrix using a capture reagent, and determining the amount of the labeled product. In this method, the AR affinity-labeled product acts as a capture reagent to capture the sample, and the method also includes steps of capturing the affinity-labeled product with the capture reagent and desorbing and ionizing the affinity-labeled product. This method involves numerous and time-consuming steps, requires specific capture reagents and multiple washing steps, and is not suitable for analyzing small molecules such as amino acids in practical applications.

[0006] Therefore, the accuracy and efficiency of amino acid detection in biological samples remains an important issue that needs to be addressed. Summary of the Invention

[0007] The main technical problem solved by this invention is to provide a method for detecting amino acids, which can detect amino acids in various biological samples. The method is accurate, easy to operate, and highly sensitive.

[0008] The present invention also provides a kit for amino acid detection.

[0009] To address the aforementioned technical problems, in a first aspect, the present invention provides a method for detecting amino acids, comprising the steps of:

[0010] Using a carboxyl-terminated long-chain polymer-biotin conjugate as a derivatization reagent, amino acid components in biological samples are derivatized to form amino acid derivatives.

[0011] The derivatization reagent used, the carboxyl-terminated long-chain polymer-biotin conjugate, is a compound represented by Formula I or Formula II, or a compound represented by Formula I or Formula II whose terminal carboxyl group is further activated by succinimidyl or a succinimidyl analogue.

[0012]

[0013] In Equation I, m takes the integer value between 2 and 6;

[0014] In Formula II, n takes the integer value between 20 and 30.

[0015] The derivatization reagent used in this invention is a carboxyl-terminated long-chain polymer-biotin conjugate (BPC), where one end of the molecule is a carboxyl group and the other end is biotin, with a polymer spacer group between the carboxyl group and the biotin. Preferably, the carboxyl terminus of the BPC is pre-activated with succinimide (NHS) or an NHS analog (such as "sulfonated NHS").

[0016] The molecular weight of the BPC can be varied as needed by changing the chain length of the polymer molecules. Specifically, the value of m or n in the structural formula can be selected to choose a specific molecular weight of "carboxyl-terminated long-chain polymer-biotin conjugate" (BPC) as a derivatizing agent to derivatize amino acid components. Preferably, the molecular weight of the BPC is in the range of 100–3000 Da.

[0017] When using biotin (BPC) as a derivatization reagent to derivatize amino acid components, BPC can react with the amino groups on the amino groups of the amino acids through activated carboxyl groups, coupling them to form a high-molecular-weight structural tag. This allows for the enhancement of the amino acid mass spectrometry signal from the low molecular weight range (m / z 50-250) to the desired molecular weight range when using MALDIMS for detection. The desired molecular weight range can be determined based on factors such as the matrix or sample type used during detection, and the presence of interfering substances. Furthermore, because both biotin and polymer chain structures contain numerous easily ionized structural sites, derivatized amino acids readily form [M+H] tags in the mass spectrometer. + or[M+Na] + Plasma adducts increase the probability and efficiency of ionization, which can greatly improve the sensitivity of mass spectrometry in detecting target signals.

[0018] As a preferred embodiment of the present invention, the present invention is mainly used to detect free amino acid components in biological samples. By derivatizing the free amino acids, the free amino acid components in the biological sample are formed into amino acid derivatives, which are then detected.

[0019] In a preferred embodiment of the present invention, the carboxyl-terminated long-chain polymer-biotin conjugate is a compound represented by Formula III or Formula IV:

[0020]

[0021] In Equation III, m takes the integer value between 2 and 6;

[0022] In Equation IV, n takes the integer value between 20 and 30.

[0023] In a preferred embodiment of the present invention, the molecular weight range of the carboxyl-terminated long-chain polymer-biotin conjugate is 100 to 3000 Da.

[0024] As a preferred embodiment of the present invention, the compound represented by Formula III is selected from N-[6-(Biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (Biotin-2AH-NHS, abbreviated as B2AN).

[0025] The structural formula of N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (B2AN) is as follows:

[0026]

[0027] Using N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester as the derivatization reagent, the molecular weight of amino acids can be increased by approximately 452 through derivatization with this molecule. This results in the mass spectrometry signal of the amino acid mainly appearing in the m / z 500-700 range, unaffected by the MALDI matrix molecule signal (signal interference from small molecule matrices mainly occurs in the m / z 100-400 range). Furthermore, due to the numerous polar groups and easily ionized sites in the B2AN structure, the derivatized molecular structure is more easily ionized than the underived amino acid molecule, improving ionization efficiency and mass spectrometry detection sensitivity. Therefore, amino acids in complex samples can be directly detected without prior purification and separation.

[0028] As a preferred embodiment of the present invention, the compound represented by Formula IV is selected from NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugate (Biotin-PEG24-NHS, abbreviated as BP24N).

[0029] The structural formula of the NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugate (BP24N) is as follows:

[0030]

[0031] Using NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugate as a derivatization reagent, amino acids can be derivatized with this molecule, increasing their molecular weight by approximately 1375. This results in the amino acid mass spectrometry signal primarily appearing in the m / z 1400-1700 range, which is not only unaffected by MALDI matrix molecule signals (signal interference from small molecule matrices mainly occurs in the m / z 100-400 range), but also less susceptible to interference from other common, highly abundant small molecules in the sample matrix (such as phospholipids), significantly improving detection accuracy. Similarly, due to the numerous polar groups and easily ionized sites in the BP24N structure, the derivatized molecular structure is more easily ionized than the underrived amino acid molecule, improving ionization efficiency and mass spectrometry detection sensitivity.

[0032] In a preferred embodiment of the present invention, during the derivatization process, the derivatization reagent is first prepared into a solution for use, and the concentration of the derivatization reagent in the prepared solution is 0.1 mM to 100 mM.

[0033] Preferably, the reaction temperature during derivatization is 20℃ to 40℃. The preferred reaction time is 5 min to 120 min.

[0034] In a preferred embodiment of the present invention, the detection method further includes the step of detecting the amino acid derivative using MALDI MS.

[0035] Furthermore, the detection method of the present invention also includes the step of purifying the biological sample before derivatization, preferably by removing large protein molecules. Purification reduces interfering substances and further improves the accuracy of detection. Of course, the biological sample can also be directly derivatized without undergoing purification to remove large protein molecules.

[0036] Preferably, methods for removing or reducing substances that interfere with MALDI MS detection, such as high-abundance proteins and salts in the sample, include organic solvent precipitation, strong acid or ultrafiltration centrifugation, etc.

[0037] Among them, the organic solvent precipitation method can use organic solvents such as methanol or acetonitrile to remove proteins from plasma samples.

[0038] In one specific implementation, the method used to remove large protein molecules is the methanol precipitation method.

[0039] A specific procedure includes: taking a biological sample, adding methanol and vortexing to precipitate the protein, then centrifuging to separate the protein and taking the supernatant.

[0040] In one specific implementation, the method used to remove large protein molecules is ultrafiltration centrifugation.

[0041] One specific operating procedure includes: taking a biological sample, adding MES buffer solution and mixing evenly, adding the mixture to an ultrafiltration centrifuge tube for centrifugation filtration (10000g / 20min) to remove high molecular weight proteins from the sample, and collecting the plasma ultrafiltrate in the lower layer of the ultrafiltration tube.

[0042] Furthermore, the detection method of the present invention also includes the step of purifying or desalting the biological sample after derivatization. Of course, the derivatized sample can also be directly detected using MALDI MS without any further processing.

[0043] In a preferred embodiment of the present invention, the detection is performed using an internal standard method for quantitative detection, and the internal standard used is an isotope-labeled amino acid.

[0044] In some embodiments, the internal standard used is an isotopically labeled amino acid. The labeling isotope of the internal standard can be selected from, for example... 15 N, Deuterium (D) 13 C isotopes.

[0045] Typically, the heavy isotopes contained in isotopically labeled amino acids include more than one heavy isotope. It is preferable to incorporate a higher number of heavy isotopes because it provides greater mass transfer. Such heavy isotopically labeled compounds are well known in the art and are available from various manufacturers.

[0046] During the testing process, the testing procedure was carried out according to the internal standard method.

[0047] In some embodiments, isotopically labeled amino acids, serving as internal standards, are added to the biological sample or standard solution prior to the derivatization reaction. These internal standards facilitate the quantitative analysis of amino acids in the sample and can also calibrate or correct inaccuracies caused by loss of the target analyte or matrix effects of interfering substances during the experiment.

[0048] In some implementations, a standard curve is plotted based on the MS signal-to-noise ratio of the amino acid standard and the internal standard and the concentration of the added amino acid, and the concentration of the amino acid in the biological sample is calculated.

[0049] In some embodiments, the amino acid derivatives are analyzed by MALDI-MS during detection, for example, using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry is obtained by accumulating and averaging 400 shots in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing are performed using Shimadzu Biotech Launchpad software (version 2.9), but other commercially available MALDI-MS mass spectrometers can also be used; this disclosure is not limiting.

[0050] Secondly, the present invention also provides an amino acid detection kit for the detection method, comprising the carboxyl-terminated long-chain polymer-biotin conjugate derivatization reagent.

[0051] In a preferred embodiment of the present invention, the kit further includes at least one of components i) to iii):

[0052] i) Reagents and consumables required for amino acid extraction and / or purification from biological samples;

[0053] ii) Reagents and consumables used in mass spectrometry detection;

[0054] iii) The isotopically labeled amino acids and amino acid standards for quantitative detection.

[0055] Thirdly, the present invention also provides the application of the detection method and the kit described herein in detecting the content of amino acid components in biological samples containing amino acids, particularly in detecting the content of free amino acid components in biological samples.

[0056] In a preferred embodiment of the present invention, the amino acid-containing biological sample is any one of an amino acid solution, dried blood smear, blood, serum, plasma, urine, saliva, and tissue fluid. Preferably, the biological sample is a blood-based sample. The blood-based sample includes dried blood smear, blood, serum, or plasma.

[0057] In some embodiments, the biological sample is a dried blood smear sample. A dried blood smear is a whole blood sample including blood cells.

[0058] The amino acids that can be detected by this invention are any one type of amino acid, such as glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, and pyrrolidone.

[0059] The method for detecting amino acids provided by this invention has the following beneficial effects:

[0060] 1) This invention completely solves the main problems existing in amino acid detection using MALDI MS. Existing amino acid derivatization methods are mostly developed for chromatographic, spectroscopic, or GC-MS, LC-MS, and cannot specifically address the problems in amino acid detection using MALDI MS. The amino acid derivatization method provided by this invention specifically solves the problems of matrix and background interference, low amino acid ionization efficiency, etc., existing in MALDI MS detection. It only requires the most classic MALDI MS procedure and a common MALDI matrix to achieve direct detection of amino acids in complex samples, without the need for additional matrix modification or the use of capture reagents to capture the derivatization reagent and / or sample. The reaction time is short, the operation steps are few, and sample loss or dilution caused by multiple washings is avoided, demonstrating extremely high practical potential.

[0061] 2) Sample preparation is simple and straightforward, the method is convenient, the instrument run time is short, and the degree of automation is high. Existing GC-MS and LC-MS methods require complex method establishment, optimization, and validation processes, as well as strong background knowledge in chromatography and mass spectrometry and professional operators. Many clinical institutions that need to determine amino acids in biological samples do not have such resources. The method provided by this invention combines BPC derivatization with MALDI MS, improving ionization efficiency and enhancing the sensitivity of MALDI-MS detection. Furthermore, it requires no chromatographic separation or enrichment processes, has a short operation time, and easily enables high-throughput detection of biological samples.

[0062] 3) Appropriate molecular weight BPCs can be selected as derivatization reagents as needed. Since different samples have different background matrices that interfere with amino acid detection, the molecular weight of the amino acid derivative can be altered by adjusting the number of polymer spacer groups in the BPC, thereby adjusting the target signal to a specific molecular weight region with less background interference. Therefore, due to their structural flexibility, these derivatization reagents are suitable for different sample types.

[0063] This invention presents the first application of a BPC-derived MALDI-MS method for the qualitative and quantitative detection of amino acids in complex biological samples. The method exhibits high specificity, accuracy, ease of operation, and high sensitivity. Experimental results demonstrate that this method can be used clinically to determine amino acids in complex biological samples. Attached Figure Description

[0064] Figure 1 This is the mass spectrometry signal spectrum of the lysine standard in Example 2 of the present invention after B2AN derivatization, detected by MALDI MS.

[0065] Figure 2This is the mass spectrometry signal spectrum of the arginine standard in Example 2 of the present invention after B2AN derivatization, detected by MALDI MS.

[0066] Figure 3 This is the mass spectrum of the plasma sample detected in Example 3 of the present invention;

[0067] Figure 4 This is the mass spectrum of the dried blood smear sample in Example 4 of the present invention;

[0068] Figure 5 This is the mass spectrum of the dried blood smear sample in Example 5 of the present invention;

[0069] Figure 6 This is a quantitative standard curve of different amino acids in Example 6 of the present invention;

[0070] Figure 7 These are the mass spectra of plasma samples from Example 7 of this invention, obtained by B2AN derivatization and without derivatization.

[0071] Figure 8 This is the mass spectrum of plasma samples derived from BPC molecules of three different molecular weights in Example 8 of the present invention. Detailed Implementation

[0072] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0073] Therefore, this invention is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in or will be apparent from the following detailed description. It will be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention.

[0074] In the following embodiments, the present invention used N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (B2AN) or NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugate (BP24N) as derivatization reagents to detect the amino acid content in several biological samples. The detection method employed an internal standard method for quantitative detection, using isotopically labeled amino acids as the internal standard.

[0075] First, the amino acids in the biological sample were derivatized to obtain amino acid derivatives. The concentration of the derivatization reagent used in the derivatization process was 0.1 mM to 100 mM. The reaction temperature for the derivatization process was 20℃ to 40℃.

[0076] The amino acid derivatives were then detected using MALDI MS.

[0077] Preferably, the biological sample is purified prior to derivatization, for example, the purification process includes purification to remove large protein molecules.

[0078] Preferably, after derivatization, the derivatized biological sample is purified or desalted.

[0079] The technical solution of the present invention will be described in detail below through specific embodiments.

[0080] Example 1

[0081] This embodiment provides a method for detecting free amino acids in plasma samples. The specific operation steps are as follows:

[0082] 1) Take 30 μL of plasma sample, add 200 μL of methanol, vortex thoroughly to precipitate proteins, centrifuge, collect the supernatant and place it in a new EP tube, then vacuum dry. Add 20 μL of pH 8.0 MES buffer solution and mix well to obtain the processed plasma sample.

[0083] B2AN was dissolved in an acetonitrile / H2O (1:1, v / v) mixed solution to prepare a B2AN solution with a concentration of 20 mM.

[0084] 2) Take 18 μL of the above-processed plasma sample and blank MES buffer solution, add 2 μL of B2AN solution to each, and react at room temperature for 30 minutes.

[0085] 3) Take 5 μL of the reaction solution obtained in step 2) after the reaction is complete, and then treat the reaction solution as follows:

[0086] Dilute 5 times with a mixture of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v);

[0087] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0088] A 1.5 μL sample was taken and spotted onto the surface of a 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.

[0089] MALDI mass spectrometry was obtained by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9).

[0090] Example 2

[0091] This example investigated the feasibility of using N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (B2AN)-derived amino acid standards. Taking arginine and lysine as examples, the steps are as follows:

[0092] 1) Prepare 100 μM solutions of arginine and lysine standards using MES buffer solution at pH 8.0. Dissolve B2AN in an acetonitrile / H2O (volume ratio 1:1) mixture to prepare a 20 mM B2AN concentration.

[0093] 2) Take 18 μL of arginine and lysine standard solutions and blank MES buffer solution, respectively, and add 2 μL of B2AN solution to each. Then react at room temperature for 30 minutes. The derivatization reaction equations of lysine and arginine with B2AN are as follows:

[0094]

[0095] The molecular weights of lysine and arginine are 146.11 and 174.11, respectively. After B2AN derivatization, the molecular weights of the detected molecules are 598.35 and 626.36, respectively. Therefore, the mass spectrometry signals of the two molecules will appear in the range above m / z 500. Within this range, the signal of the detected molecules will not be interfered with by the background signal of the MALDI matrix (the background signal interference of commonly used MALDI matrices such as dihydroxybenzoic acid usually appears in the range of m / z 100-400).

[0096] 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);

[0097] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0098] 1.5 μL of the sample solution was spotted onto a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The analytical results are shown below. Figure 1 and Figure 2 As shown. Among them, Figure 1 The mass spectrometry signal of lysine standard after B2AN derivatization, detected by MALDI MS, and the comparison spectrum with the blank sample are shown. Figure 2 The mass spectrometry signal of arginine standard after B2AN derivatization was detected by MALDI MS and the comparison spectrum with the blank sample.

[0099] Figure 1 and Figure 2 This indicates that MALDI MS can detect the B2AN derivative signals of arginine and lysine with high sensitivity. Conversely, the blank sample showed no significant interference peaks in this signal region. This result demonstrates that it is feasible to derivatize amino acids with B2AN and utilize the signals of B2AN derivatives for the detection and quantification of amino acids. B2AN derivatization can increase the molecular weight of amino acids to above m / z 500, within which there is virtually no matrix signal interference. This significantly improves the sensitivity and ionization efficiency of amino acid detection, achieving high-sensitivity detection with MALDI MS, free from matrix background interference in the detection region, and eliminating the need for complex chromatographic separation and purification.

[0100] Example 3

[0101] This embodiment provides a method for detecting free amino acids in plasma samples, using B2AN derivatization combined with MALDI MS detection. The specific operation steps are as follows:

[0102] 1) Take 60 μL of plasma sample and add an equal volume of pH 8.0 MES buffer solution and mix well. Add the mixture to a 3 kDa ultrafiltration centrifuge tube and centrifuge (10000 g / 20 min) to remove high molecular weight proteins from the sample. Collect the plasma ultrafiltrate in the lower layer of the ultrafiltration tube into a new EP tube.

[0103] Dissolve B2AN in an acetonitrile / H2O (volume ratio 1:1) mixed solution to prepare a B2AN concentration of 20 mM.

[0104] 2) Take 18 μL of the above plasma ultrafiltrate and blank MES buffer solution, add 2 μL of B2AN solution to each, and react at room temperature for 30 minutes.

[0105] 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);

[0106] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0107] 1.5 μL of the sample solution was spotted onto a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 3 As shown. Figure 3 (a) shows the mass spectrum of free amino acids in a plasma sample after ultrafiltration and centrifugation to remove high-abundance proteins, followed by B2AN derivatization and analysis using MALDI MS. Figure 3 (b) is the mass spectrum of the blank control sample.

[0108] Depend on Figure 3 It is known that most amino acid molecules in blood plasma are in the [M+H] group. + or[M+Na] +The ions were detected and labeled in the figure. In contrast, no corresponding signal was observed in the blank sample. In this study, free amino acids in plasma were directly and sensitively detected by MALDI MS via a BPC molecule derivatization reaction (B2AN in this figure), without any complex chromatographic separation and purification steps.

[0109] By derivatizing amino acids with B2AN, MALDI MS can directly detect different types of amino acid signals in plasma samples without any chromatographic separation or purification process; the entire procedure is simple and rapid. This result demonstrates that it is feasible to derivatize amino acids with B2AN and utilize the signals of the B2AN derivatives to detect amino acid components in complex sample matrices. B2AN derivatization can increase the molecular weight of amino acids to above m / z 500, a range largely free from matrix signal interference, significantly improving the sensitivity and ionization efficiency of amino acid detection.

[0110] Example 4

[0111] This embodiment provides a method for detecting amino acids in dried blood smear samples. The specific operation steps are as follows:

[0112] 1) Separate the 5mm diameter blood smear sample from the dried blood smear using a perforation method and place it in an EP tube. Add 100μL of methanol to the tube, vortex for 10 minutes, remove the methanol supernatant and place it in a new EP tube, then vacuum dry the solvent. Simultaneously prepare the corresponding blank paper sample.

[0113] B2AN was dissolved in a 1:1 mixture of acetonitrile and H2O to prepare a 20 mM solution.

[0114] 2) Add 18 μL of MES buffer solution (pH 8.0) to the above dried blood smear extract sample tube, vortex thoroughly, then add 2 μL of B2AN solution and mix well. React at room temperature for 30 minutes.

[0115] 3) Take 5 μL of the reaction mixture obtained in step 2) and dilute it 5 times with a mixture of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v);

[0116] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0117] 1.5 μL of the sample solution was spotted onto a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). Results are as follows: Figure 4 As shown. Figure 4 Image (a) shows the mass spectrum of a dried blood smear sample after methanol extraction, derivatization with B2AN, and detection of free amino acids by MALDI MS. Figure 4 (b) is the mass spectrum of the blank control sample.

[0118] Depend on Figure 4 It can be seen that most amino acid molecules in dried blood smears are in the [M+H] group. + or[M+Na] + Ions were detected and labeled in the figure. In contrast, no corresponding signal was observed in the blank sample. In this study, free amino acids in dried blood smears were directly and sensitively detected by MALDI MS via a BPC molecule derivatization reaction (B2AN in this figure), without any complex chromatographic separation and purification steps.

[0119] By derivatizing amino acids with B2AN, MALDI MS can directly detect different types of amino acid signals in dried blood smear samples without any chromatographic separation or purification process. The entire procedure is simple and rapid. This result demonstrates that it is feasible to use B2AN to derivatize amino acids and utilize the signals of B2AN derivatives to detect amino acid components in different types of complex sample matrices, with a high signal-to-noise ratio and good detection sensitivity.

[0120] Example 5

[0121] To investigate the derivatization and detection effects of carboxylated polyethylene glycol-biotin conjugates (BPCs) of different molecular weights on amino acids, this example specifically tested the feasibility of using NHS-activated carboxylated polyethylene glycol-biotin conjugate (BP24N) to derivatize and detect amino acids in dried blood smear samples. The specific operating steps are as follows:

[0122] 1) Separate the 5mm diameter blood smear sample from the dried blood smear using a perforation method, place it in an EP tube, add 100μL of methanol to the tube, vortex for 10 minutes, remove the methanol supernatant and place it in a new EP tube, and vacuum dry the solvent. At the same time, prepare the corresponding blank paper sample.

[0123] Dissolve BP24N in pure water to prepare a concentration of 20 mM.

[0124] 2) Add 18 μL of MES buffer solution at pH 8.0 to the above dried blood smear extract sample tube, vortex thoroughly, then add 2 μL of BP24N solution and mix well. React at room temperature for 30 minutes.

[0125] 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);

[0126] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0127] 1.5 μL of the sample solution was spotted onto the surface of a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 5 As shown. Figure 5 Image (a) shows the mass spectrum of dried blood smear samples after methanol extraction, derivatization with NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugate (BP24N), and detection of free amino acids by MALDI MS. Figure 5 (b) shows the results for the blank control sample.

[0128] Most amino acid molecules in dried blood slices are composed of [M+K]. + or[M+Na] + Ions were detected and labeled in the figure. In contrast, no corresponding signal was observed in the blank sample. In this study, free amino acids in dried blood smears were directly and sensitively detected by MALDI MS via a BPC molecule derivatization reaction (BP24N in this figure), without any complex chromatographic separation and purification steps.

[0129] By using BP24N derivatization, MALDI MS can directly detect different amino acid signals in dried blood smear samples without any chromatographic separation or purification process; the entire procedure is simple and rapid. This result also demonstrates that it is feasible to derivatize and detect amino acid components in complex sample matrices using carboxylated polyethylene glycol-biotin conjugates (BPCs) of different molecular weights. Depending on factors such as the type of sample matrix and the molecular weight of interfering substances, an appropriate molecular weight BPC can be selected to derivatize amino acids, shifting the amino acid signal to a less interfering molecular weight range to obtain the best detection sensitivity and specificity.

[0130] Example 6

[0131] This embodiment examines the feasibility of using B2AN derivatization combined with MALDI MS detection for the quantitative analysis of free amino acids in samples and the determination of its standard curve. The steps are as follows:

[0132] 1) Prepare a 25 μM lysine isotope internal standard solution using MES buffer solution at pH 8.0. This lysine isotope internal standard contains six... 13 C isotopes. This internal standard buffer solution was used to dilute and prepare mixed standard solutions of amino acids at different concentrations. These mixed standard solutions contained different types of amino acid standards, including glycine, alanine, threonine, proline, lysine, histidine, phenylalanine, and arginine. The concentration of each amino acid standard varied slightly, with the overall concentration ranging from 0.1 to 80 μM.

[0133] B2AN was dissolved in an acetonitrile / H2O (1:1) mixed solution to prepare a concentration of 20 mM.

[0134] 2) Take 18 μL of the above amino acid mixed standard solution and add 2 μL of B2AN solution to each solution, and react at room temperature for 30 minutes.

[0135] 3) Take 5 μL of the reaction mixture and dilute it 5 times with a mixture of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v);

[0136] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0137] 1.5 μL of the sample solution was spotted onto a stainless steel target plate, and the amino acid derivatives were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). All spectra were normalized using a lysine isotope internal standard. A quantitative standard curve for amino acids was plotted with the concentration of amino acid standards on the x-axis and the mass spectrometric response of the amino acid derivative signal on the y-axis. The results are shown below. Figure 6 As shown. Figure 6 The standard curves for the quantitative analysis of amino acids using the method described in this invention are shown. A mixed standard solution of eight amino acids was tested, with an isotopic standard of lysine used as an internal standard. The solution contained different types of amino acid standards, including glycine, alanine, threonine, proline, lysine, histidine, phenylalanine, and arginine. The concentrations of each amino acid standard varied slightly, with an overall concentration range of 0.1–80 μM. The results show that a suitable standard curve for amino acid quantification can be obtained by combining B2AN and MALDI MS detection. The R-values ​​of all amino acid standard curves are [not specified in the original text]. 2 All values ​​were above 0.99, and the response values ​​at each concentration showed good reproducibility, with CV% all below 20%, which meets the requirements for quantitative analysis of amino acids.

[0138] Example 7

[0139] This example compares the performance of MALDI MS in detecting plasma amino acids under two conditions: derivatization with BPC (using B2AN as the derivatization reagent) and no derivatization. The specific operating steps are as follows:

[0140] 1) Take two plasma samples, 30 μL of plasma in each. Add 200 μL of methanol to each sample to precipitate high molecular weight proteins. Shake the mixture for 2 minutes, then centrifuge at 10000g for 20 minutes. Transfer the supernatant from the sample tubes to two new EP tubes. Then vacuum dry the solvent in both tubes.

[0141] Dissolve B2AN in an acetonitrile / H2O (volume ratio 1:1) mixed solution to prepare a B2AN concentration of 20 mM.

[0142] 2) Add 18 μL of MES buffer solution (pH 8.0) to both sample tubes. Vortex thoroughly. Then, add 2 μL of B2AN solution to one sample tube and mix well. Incubate at room temperature for 30 minutes. Add 2 μL of blank aqueous solution to the other sample tube.

[0143] 3) Take 5 μL of each of the two reaction mixtures obtained in step 2) and dilute them 5 times with a mixture of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v);

[0144] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0145] 1.5 μL of the sample solution was spotted onto a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 7 As shown. Figure 7 Image (a) shows the mass spectrum of free amino acids after B2AN derivatization, detected by MALDI MS. Figure 7 (b) shows the mass spectrum of the corresponding molecular weight region of amino acids detected by MALDI MS without any derivatization (theoretically, the signal of underived amino acids is in the range of m / z 50-300).

[0146] Depend on Figure 7 It is known that BPC derivatization and MALDI MS detection can reliably obtain highly sensitive plasma free amino acid signals; conversely, without derivatization, no reliable amino acid signals can be detected by MALDI MS in the same sample, and the main signals in the mass spectrometer come from background components such as matrix molecules (e.g., DHB).

[0147] Example 8

[0148] This example compares the performance of MALDI MS in detecting plasma amino acids using three BPC molecules with different spacer chain lengths and molecular weights. The three BPC molecules are: N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (B2AN, molecular weight 567.7 Da), N-succinimide-6-biotinaminohexanoic acid (B1AN, molecular weight 454.5 Da), and D-biotin N-hydroxysuccinimide ester (BOAN, molecular weight 341.4 Da). The specific operating steps are as follows:

[0149] 1) Take three plasma samples, 30 μL of plasma in each sample. Add 200 μL of methanol to two of the samples to precipitate high molecular weight proteins. Shake the mixture for 2 minutes, then centrifuge at 10000g for 20 minutes. Transfer the supernatant from the sample tubes into three new EP tubes. Then vacuum dry the solvent in the three tubes.

[0150] B2AN, B1AN, and B0AN were dissolved in a acetonitrile / H2O (volume ratio 1:1) mixed solution, each with a concentration of 20 mM.

[0151] 2) Add 18 μL of MES buffer solution with pH 8.0 to each of the three sample tubes. After vortexing thoroughly, add 2 μL of B2AN, B1AN, and B0AN solutions to each of the three sample tubes and mix well. React at room temperature for 30 minutes.

[0152] 3) Take 5 μL of each of the three reaction mixtures obtained in step 2) and dilute them 5 times with a mixture of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v);

[0153] Take 1.5 μL of the diluent and mix it with 1.5 μL of 2,5-dihydroxybenzoic acid solution to prepare the test sample. The 2,5-dihydroxybenzoic acid solution is an acetonitrile / water / trifluoroacetic acid solution of 2,5-dihydroxybenzoic acid (1:1:0.002, v / v / v) with a concentration of 10 mg / mL.

[0154] 1.5 μL of the sample solution was spotted onto a 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. MALDI mass spectrometry was performed by accumulating and averaging 400 shots in cation mode with a laser intensity of 100-120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 8 As shown. Figure 8(a), (b), and (c) show the mass spectra of free amino acids after derivatization with B2AN, B1AN, and B0AN, respectively, detected by MALDI MS.

[0155] Depend on Figure 8 It was found that derivatization with BPC of different molecular weights could collect certain plasma free amino acid signals. However, we also observed that the number of detectable amino acid signals decreased significantly with decreasing BPC molecular weight, especially with B0AN at a molecular weight of 341, where only two or three amino acid signals with high signal-to-noise ratios could be detected after derivatization. This is because plasma samples exhibit significant background interference in the m / z 300-500 range. These signals originate from both high-abundance substances such as lipids and salts in the plasma and background peaks in the MALDI mass spectrometry matrix. Since B0AN 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 indicates that to obtain ideal detection results, the molecular weight of the amino acid derivatization reagent cannot be too small, and the derivatization reaction must be able to elevate the amino acid detection signal to at least the range above m / z 500 (such as B2AN) to achieve a relatively reliable and highly sensitive analysis. Previously developed amino acid derivatization reagents mostly have molecular weights below 300 Da and are not suitable for MALDI MS detection of complex samples.

[0156] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0157] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting amino acids, characterized in that, Including the following steps: Using a carboxyl-terminated long-chain polymer-biotin conjugate as a derivatization reagent, amino acid components in biological samples are derivatized to form amino acid derivatives; and The amino acid derivatives were detected using MALDI MS; The carboxyl-terminated long-chain polymer-biotin conjugate is a compound represented by Formula I, Formula II, Formula III, or Formula IV: ； Formula I ； Formula II ； Formula III ； Formula IV In Equation I, m takes the integer value between 2 and 6; In Formula II, n takes the integer value between 20 and 30; In Equation III, m takes the integer value between 2 and 6; In Equation IV, n takes the integer value between 20 and 30.

2. The detection method according to claim 1, characterized in that, The molecular weight range of the carboxyl-terminated long-chain polymer-biotin conjugate is 100–3000 Da.

3. The detection method according to claim 1, characterized in that, The compound represented by Formula III is selected from N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester; and / or, the compound represented by Formula IV is selected from NHS-activated carboxylated poly(24-ethylene glycol)-biotin conjugates.

4. The detection method according to any one of claims 1 to 3, characterized in that, When performing the derivatization process, the concentration of the derivatization reagent used is 0.1 mM to 100 mM.

5. The detection method according to any one of claims 1 to 3, characterized in that, The reaction temperature for the derivatization treatment is 20℃~40℃.

6. The detection method according to any one of claims 1 to 3, characterized in that, The detection method further includes the following steps: The biological sample is purified before the derivatization process.

7. The detection method according to claim 6, characterized in that, The purification process is a purification process to remove large protein molecules.

8. The detection method according to claim 6, characterized in that, Following the derivatization process, the biological sample is purified or desalted.

9. The detection method according to any one of claims 1 to 3, characterized in that, The detection method employs internal standard method for quantitative detection, and the internal standard used is an isotope-labeled amino acid.

10. A kit for amino acid detection used in the detection method according to any one of claims 1 to 9, characterized in that, This includes the carboxyl-terminated long-chain polymer-biotin conjugate.

11. The reagent kit according to claim 10, characterized in that, The kit also 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 in mass spectrometry detection; iii) Isotope-labeled amino acids and amino acid standards for quantitative detection.

12. The application of the detection method according to any one of claims 1 to 9 or the kit according to claim 10 or 11 in detecting the content of amino acid components in biological samples.

13. The application according to claim 12, characterized in that, The biological sample can be any one of amino acid solution, dried blood smear, blood, serum, plasma, urine, or saliva.

14. The application according to claim 12, characterized in that, The biological sample is tissue fluid.

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