Method for screening and identifying hexose saccharification product based on tandem mass spectrometry

Abstract

The invention discloses a method for screening and identifying a hexose saccharification product based on tandem mass spectrometry. According to the method, two specific neutral losses in MS / MS of the polysaccharide saccharification product are analyzed, so that efficient, accurate and reliable identification of the polysaccharide saccharification product is realized. The method mainly comprises the following steps: screening potential hexose saccharification products by using neutral loss 180.063, identifying hexose and precursor compounds by using neutral loss 162.052 and 144.042, and finally completing identification of the hexose saccharification products. The method disclosed by the invention has the characteristics of high efficiency, accuracy and reliability, is suitable for the fields of biochemistry, food chemistry analysis and the like, and provides an effective solution for rapid screening and identification of hexose saccharification products.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for screening and identifying hexasaccharide glycation products based on tandem mass spectrometry. Background Art

[0002] Hexasaccharide is a polysaccharide compound formed by dehydration condensation of two or more hexose molecules and widely exists in various living organisms. Hexasaccharide can have different molecular weights ranging from several hundred to tens of thousands of daltons. The most common hexasaccharides include lactose, maltose, sucrose, starch, cellulose, etc.

[0003] The Mailard reaction refers to the reaction in which the aldehyde or ketone group of a reducing sugar condenses and rearranges with a free amino group to form a series of compounds. Glycation products (Amadori / Heyns products) are stable compounds formed in the Mailard reaction. Glycation products also play a key role in food processing and food quality control. Substances containing hexasaccharide or other polysaccharides, such as dairy products and desserts, can generate glycation products when reacting with other amino compounds during heating and processing.

[0004] Currently, there is no specific screening and identification method for hexasaccharide glycation products at home and abroad. Summary of the Invention

[0005] To solve the disadvantages and deficiencies of the prior art, the present invention provides a method for screening and identifying hexasaccharide glycation products based on tandem mass spectrometry.

[0006] The present invention uses liquid chromatography-mass spectrometry technology to detect hexasaccharide glycation products and rapidly screens and identifies hexasaccharide glycation products by the method of neutral loss (162.052, 180.063, 144.042) screening.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A method for screening and identifying hexasaccharide glycation products based on tandem mass spectrometry, comprising the following steps:

[0009] (1) Detection of the sample to be tested: The sample to be tested is detected by chromatography-mass spectrometry to obtain the primary and secondary mass spectrometry information data of the sample (this step aims to obtain mass spectrometry data related to hexasaccharide glycation products);

[0010] (2) Analysis of the original data: The original data file collected in step (1) is subjected to extraction of the primary mass spectrometry peaks and corresponding secondary fragments to obtain the ion addition situation and mass-to-charge ratio of the substance (this helps to screen potential hexasaccharide glycation products in the subsequent steps);

[0011] (3) Screening for potential hexasaccharide glycosylation products: Taking single-charged ions as an example, substances with a charge state of usually [M+H] + / [M+Na] + / [M+K] + are screened (hereinafter denoted as [M+X] + ). Then, the ion fragments in the secondary mass spectrum are first screened using a neutral loss of 180.063 (C6H12O6). If the corresponding ion fragments are found, the substance corresponding to the spectrum is denoted as a potential hexasaccharide glycosylation product;

[0012] If there is no fragment with a neutral loss of 180.063 in the secondary spectrum, continue to search for fragments with a neutral loss of 162.052 (C6H10O5); if 162.052 fragments are found, the substance is identified as a monohexose glycosylation product; if neither 180.063 fragments nor 162.052 fragments are found, the substance is classified as a non-hexose glycosylation product;

[0013] (4) Continuing to identify hexasaccharide glycosylation products from the secondary spectra after the first screening: For substances that are potential hexasaccharide glycosylation products, the secondary ion fragments are screened using a neutral loss of 162.052; in the secondary spectrum, find the fragment ion [M+X-180.063] + where the parent ion loses 180.063. Starting from this ion, search for ion fragments with a neutral loss of 162.052 in the low-molecular-weight fragment ions one by one from high to low in the secondary spectrum until there is no longer a 162.052 neutral loss, denoted as [M+X-180.063-n×162.052] + (n≥0). Then, subtract the neutral loss of 144.042 (C6H8O4) from the m / z value of this fragment ion to obtain the mass-to-charge ratio m P / z of the precursor compound; identify this m P / z to determine the precursor substance of the glycosylation product, and this substance is identified as (n + 2) hexasaccharide glycosylation products (n≥0).

[0014] Furthermore, the mass-to-charge ratio m P / z of the precursor compound obtained in step (4) is then used to search the database or collect its secondary mass spectrum for identification to complete the identification of the precursor compound of the hexasaccharide glycosylation product, and finally the name of the hexasaccharide glycosylation product can be determined.

[0015] Furthermore, the hexasaccharide refers to a polysaccharide formed by two or more hexoses.

[0016] Furthermore, the hexasaccharide glycosylation products may include hexasaccharide glycides and their isomers.

[0017] Furthermore, the hexosaccharide glycation products are mainly Amadori or Heyns rearrangement products (Amadori / Heyns rearrangement products).

[0018] Furthermore, the chromatography method in step (1) can be high performance liquid chromatography (HPLC), capillary electrophoresis chromatography or gas chromatography (GC).

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] The method of the present invention can accurately and rapidly screen out hexosaccharide glycation products according to the characteristic neutral loss of hexosaccharides and identify precursor compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the method for screening hexosaccharide glycation products based on mass spectrometry according to the present invention.

[0022] Figure 2 is the secondary mass spectrum of a self-made standard of glutamate glycated with cellobiose (lactose) and the change rule of neutral loss fragments under different collision energies.

[0023] Figure 3 is the secondary mass spectrum identification diagram of a self-made standard of alanine glycated with maltotriose (maltotriose).

[0024] Figure 4 is the secondary mass spectrum identification result diagram of different hexosaccharide glycated threonine products in milk powder samples.

[0025] Figure 5 is the secondary mass spectrum identification of cellobiose glycated peptides in milk powder.

[0026] Figure 6 is the chromatogram (a) and secondary mass spectrum identification (c) of phosphatidylethanolamine glycated with cellobiose (lactose) in milk powder.

[0027] Figure 7 is the distribution and PCA clustering diagram of hexosaccharide glycation products in different milk powders. In the figure, A, B, C, D, and E represent milk powders of different brands respectively.

[0028] Figure 8 is the screening process of hexosaccharide glycation products. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described in detail below with reference to the embodiments and the drawings, but the embodiments of the present invention are not limited thereto. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0030] Example 1: Preparation and MS / MS Detection of Standard Polysaccharide Glycation Products

[0031] 1. Preparation of Standard Amino Acid Polysaccharide Glycation Products

[0032] Prepare a 200 mM amino acid standard solution and 200 mM D-(+)-glucose, lactose, and maltotriose standard solutions. Mix 15 amino acid solutions with the polysaccharide solution in a volume ratio of 1:1 and dry quickly under vacuum. Subject the dried sample to a metal bath at 70 °C for 2 hours to obtain the standard amino acid glycation products.

[0033] 2. Detection of Standard Polysaccharide Glycation Products

[0034] Use Thermo Ultimate 3000 UHPLC and Q-Exactive Orbitrap MS for detection.

[0035] Chromatographic method:

[0036] Perform chromatographic separation using a ZIC-HILIC column (2.1 mm × 100 mm, 3.5 μm, Millipore, Darmstadt, Germany). The analysis conditions are as follows: Solvent A, water / formic acid (99.9:0.1, v / v), containing 20 mM ammonium acetate; Solvent B, ACN / formic acid (99.9:0.1 v / v); Gradient, initial 80% (v / v) B for 4 min, linear 80 - 20% (v / v) B for 15 min, 20% (v / v) B held for 10 min, 80% (v / v) B for column equilibration for 5 min; Flow rate: 0.2 mL / min; Injection volume, 2 μL.

[0037] Mass spectrometry method:

[0038] Data-dependent acquisition mode: In each cycle, the 10 most intense precursor ions are selected for fragmentation (TOP10); First-stage scan range: m / z 60 - 900; First-stage resolution: 70000; AGC target value: 3e6; Fragmentation mode: HCD; Fragmentation energy: 20%; Second-stage resolution: 17500; Maximum ion injection time: 100 ms;

[0039] Figure 2 This shows the ion fragmentation pattern of self-made lactosylated glutamate as a function of collision energy, indicating that glycation products can be identified by characteristic neutral loss under certain collision energy conditions. Figure 3 This is the mass spectrometry identification chart of maltotriosylated alanine, which can be accurately identified using the method of the present invention.

[0040] Example 2: Screening and Identification of Polysaccharide Glycation Products in Milk Powder

[0041] 1. Sample pretreatment:

[0042] Accurately weigh 0.3 g of milk powder sample into a 15 ml centrifuge tube, add 3 ml of room temperature deionized water, and mix well to dissolve. Add 12 ml of methanol and mix thoroughly. Place at -20 °C for 30 minutes to precipitate macromolecular proteins. After centrifugation, add chloroform for extraction, vortex mix, and let stand for 30 min to separate the layers. Centrifuge further to separate the organic phase from the aqueous phase. Carefully collect the upper layer (0.5 mL) and use rapid vacuum drying. Finally, store the dried sample at -80 °C for later use. Combine equal amounts of each sample together to form a mixed QC sample. In addition, three solvent blank samples were prepared in the same way.

[0043] 2. Sample detection

[0044] Sample detection was carried out on a Thermo Ultimate 3000 UHPLC and Q-Exactive Orbitrap MS equipped with a heated electrospray ionization source (HESI-II). Chromatographic separation was performed using a ZIC-HILIC column (2.1 mm × 100 mm, 3.5 μm, Millipore, Darmstadt, Germany). The analytical conditions were as follows: Solvent A, water / formic acid (99.9:0.1) containing 20 mM ammonium acetate; Solvent B, ACN / formic acid (99.9:0.1); Gradient: initial 80% B for 4 min, 80 - 20% B for 15 min, 20% B held for 10 min, 80% B for column equilibration for 5 min; Flow rate: 0.2 ml / min; Injection volume, 5 μL. The auto sampler and column oven temperatures were set at 6 °C and 40 °C, respectively.

[0045] For the non-targeted screening and identification of glycation products in milk powder, Full MS-ddMS 2 mode was used. The mass spectrometry analysis was carried out in the positive ion mode. The Full MS mass scan range was m / z 60 - 900. The collision energy for the ddMS2 experiment was set at NCE 25%. The mass resolution of the full mass spectrum was 70000, and that of ddMS2 was 17500.

[0046] 3. Result analysis

[0047] MS / MS data with a neutral loss of 180.063 in the secondary mass spectrum were initially screened for further identification: In the secondary spectrum, find the fragment ions with a neutral loss of 180.063 from the precursor ion. Starting from this ion, search for ion fragments with a neutral loss of 162.052052 in the low molecular weight fragment ions in the secondary spectrum until there is no such neutral loss. Then subtract 144.042 from the m / z of this fragment ion to obtain the precursor compound mass-to-charge ratio denoted as m P / z, identify the precursor compound by database retrieval according to the secondary spectrum of the precursor compound.

[0048] Figure 4 For the secondary spectra of hexosaccharide alanine of different sizes, identify the hexosaccharide glycated amino acids in milk powder according to the characteristic peaks of hexosaccharide neutral loss; Figure 5 For the secondary spectrum of the identified lactosylated peptide segment; Figure 6 For the identification display of the chromatographic peak and secondary spectrum of lactosylated phosphoethanolamine; Some of the identified hexosaccharide glycation products ([Hex]n) are shown in Table 1:

[0049] Table 1 Identification results of hexosaccharide glycation products in milk powder

[0050]

[0051]

[0052]

[0053] Use mass spectrometry peak extraction software to process the original file generated by full-scan mass analysis, extract the peak areas for identifying Amadori products, and conduct comparative analysis on the responses of hexosaccharide glycation products in different milk powders. Figure 7 For the Venn diagram and PCA diagram drawn after peak integration of the identified hexosaccharide glycation products in milk powder.

[0054] Example 3: Establishment of the screening process for hexosaccharide glycation products

[0055] Mass spectrometry data preprocessing: Perform mass spectrometry peak extraction on the mass spectrometry data collected by LC-MS / MS to obtain information such as the first-level mass spectrometry and second-level mass spectrometry of the test samples.

[0056] Edit the screening program for hexosaccharide glycation products:

[0057] Search for ion fragments with a neutral loss of 180.063 in the secondary mass spectrometry diagram, and extract the mass spectrometry diagrams with 180.063 neutral loss fragments for further screening. From [M+H-180.063] + Start searching for a neutral loss of 162.052 towards lower m / z, record the number of 162.052 fragments obtained as n, and then search for ion fragments with a neutral loss of 144.042. The mass-to-charge ratio of this fragment is the m / z of the precursor compound of the hexosaccharide glycation product. Identify this m / z according to database retrieval or other methods. Identify this compound as (n + 2) hexosaccharide compound. The principle of the screening program is as Figure 8 shown.

[0058] The neutral losses 180.063, 162.052, and 144.042 described in the present invention are theoretical values, and error adjustments should be made according to the instrument performance in actual applications.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for screening and identifying hexosaccharide glycation products based on tandem mass spectrometry, characterized in that, Including the following steps: (1) Detecting a sample to be measured by chromatography-mass spectrometry to obtain the first-level and second-level mass spectrometry data of the sample; (2) Extracting the first-level mass spectrometry peaks and corresponding second-level fragments from the original data file collected in step (1) to obtain the ion addition situation and mass-to-charge ratio of the substance; (3) Taking single-charge ions as an example, the substances with the usually charged states of [M+H] + / [M+Na] + / [M+K] + (hereinafter denoted as [M+X] + ) are screened. Then, neutral loss of 180.063 (C6H12O6) is used to conduct the first screening of the ion fragments in the secondary mass spectrum. If the corresponding ion fragments are found, the substance corresponding to this spectrum is denoted as the potential hexosan glycosylation product; If no neutral loss of 180.063 is found, the substance is classified as a non-hexosylated product; (4) For the substance of potential hexosan glycosylation product, find the fragment ion [M+X-180.063] with a 180.063 loss of the parent ion in the secondary spectrum + , starting from this ion, in the secondary spectrum from high to low, successively search for the ion fragment with a neutral loss of 162.052 (C6H10O5) among the low molecular weight fragment ions until there is no longer a 162.052 neutral loss, denoted as [M+X-180.063-n×162.052] + , where n≥0, and then subtract the neutral loss of 144.042 (C6H8O4) from the m / z value of this fragment ion to obtain the precursor compound mass-to-charge ratio denoted as m P / z; identifying this m P / z can determine the precursor substance of the glycosylation product, and this substance is identified as the (n+2) hexosan glycosylation product.

2. The method according to claim 1, characterized in that Subtracting all 180.063, 162.052, and 144.042 fragments from the (n + 2) hexosylated products screened in step (4) to obtain the mass-to-charge ratio of the precursor compound, then retrieving it using a metabolite database, and comparing the retrieval result with the second-level spectrum of the compound to complete the identification of the precursor compound of the hexosylated product, and the name of the hexosylated product can be determined.

3. The method according to claim 1, characterized in that, The hexosaccharide refers to a polysaccharide formed by two or more hexoses.

4. The method according to claim 1, characterized in that, The hexosylated products include hexosylated compounds and their isomers.

5. The method according to claim 1, characterized in that The hexosylated products are mainly Amadori or Heyns rearrangement products.

6. The method according to claim 1, wherein The chromatography method in step (1) is high-performance liquid chromatography, capillary electrophoresis chromatography or gas chromatography.

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