A method for detecting acrolein and formaldehyde exposure

Abstract

The present invention discloses a method for detecting acrolein and formaldehyde exposure, and relates to the field of biotechnology. The method comprises the following steps: performing protein reconstruction and enzymatic hydrolysis on a protein sample to be tested to obtain an enzymatic hydrolysate; performing mass spectrometry analysis on the enzymatic hydrolysate, and when a lysine residue shows an increase in molecular weight of 106.04Da, it is judged that the sample to be tested has been exposed to acrolein and formaldehyde environments at the same time. The present invention constructs an adduct of formaldehyde, acrolein and N-acetyl lysine, and analysis finds that formaldehyde and acrolein are jointly bound to the side chain amino group of lysine, which will increase the molecular structure of 106.04Da. By utilizing the increase in the molecular structure of 106.04Da of the protein lysine side chain, it can be judged whether the biological sample has been exposed to formaldehyde and acrolein environments at the same time. The present invention provides technical support for the rapid analysis of environmental exposure of acrolein and formaldehyde and acrolein and formaldehyde modifications in proteins.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting acrolein and formaldehyde exposure. Background Art

[0002] Formaldehyde (FA) and acrolein (ACR) are typical reactive carbonyl compounds (RCSs) with both endogenous and exogenous sources. FA and ACR are widely present in food, and human exposure is high on a daily basis. RCSs are extremely reactive and can attack and modify biomacromolecules such as proteins, DNA, and RNA in the body, posing a threat to human health. Regarding ACR, four ACR-induced protein adducts have been identified: Schiff bases of lysine, Michael adducts of cysteine, histidine, and lysine, N-(3-formyl-3,4-dehydropiperidinyllysine) (FDP-lysine), and N-ε-3-methylpyridinyllysine (MP-lysine). Formaldehyde, on the other hand, typically cross-links proteins via a methylene bridge mechanism. Specifically, it forms Mannich bases with free amino groups (such as lysine residues), which then undergo chemical modification through interactions with other amino groups, leading to both intramolecular and inter-molecular modifications. Due to its long-term chronic health effects, formaldehyde has been classified as a Class A carcinogen by the International Agency for Research on Cancer (IARC), with an endogenous conversion rate as high as 878-1310 mg / kg per day. Acrolein has been designated as a high-priority hazardous air pollutant by relevant authorities. Acrolein and formaldehyde are formed simultaneously in the environment, food, and the body, and the two harmful aldehydes often coexist in these exposure environments. However, research on the structures and sites of protein co-modification by acrolein and formaldehyde is currently lacking, as are methods for detecting co-exposure to acrolein and formaldehyde.

[0003] The present invention intends to develop a method for detecting acrolein and formaldehyde exposure, thereby achieving efficient detection of simultaneous acrolein and formaldehyde exposure in biological samples. Summary of the Invention

[0004] The present invention aims to provide a method for detecting acrolein and formaldehyde exposure to address the above-mentioned problems in the prior art. The present invention provides technical support for the rapid analysis of environmental exposure to acrolein and formaldehyde, as well as acrolein and formaldehyde modifications in proteins.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for detecting acrolein and formaldehyde exposure, comprising the following steps:

[0007] Perform protein reconstruction and enzymatic hydrolysis on the protein sample to be tested to obtain an enzymatic hydrolyzate;

[0008] The enzymatic hydrolysate is subjected to mass spectrometry analysis. When the molecular weight of the lysine residue increases by 106.04 Da, it is determined that the sample to be tested has been exposed to acrolein and formaldehyde environments at the same time.

[0009] Furthermore, the protein sample to be tested is placed in a PBS solution containing urea for protein reconstitution.

[0010] Furthermore, the concentration of the urea in the PBS solution is 8M.

[0011] Furthermore, the protease used in the enzymatic treatment is trypsin.

[0012] The present invention also provides a method for identifying lysine chemical modification sites of acrolein and formaldehyde, comprising the following steps:

[0013] Perform protein reconstruction and enzymatic hydrolysis on the protein sample to be tested to obtain an enzymatic hydrolyzate;

[0014] The enzymatic hydrolysate is subjected to mass spectrometry analysis. When a molecular weight increase of 106.04 Da is observed at the lysine residue site, it is determined that the lysine residue site is a lysine chemical modification site of acrolein and formaldehyde.

[0015] Furthermore, the protein sample to be tested is placed in a PBS solution containing urea for protein reconstitution.

[0016] Furthermore, the concentration of the urea in the PBS solution is 8M.

[0017] Furthermore, the protease used in the enzymatic treatment is trypsin.

[0018] The present invention also provides an adduct of formaldehyde, acrolein and N-acetyl lysine, the structural formula of which is as follows:

[0019]

[0020] The present invention also provides use of the above-mentioned adduct in detecting exposure to acrolein and formaldehyde.

[0021] The present invention discloses the following technical effects:

[0022] This study constructs adducts of formaldehyde, acrolein, and N-acetyl lysine. Analysis reveals that when formaldehyde and acrolein co-bind to the amino group on the side chain of lysine, a 106.04 Da molecular mass is added. This 106.04 Da molecular mass increase on the lysine side chain of proteins can be used to determine whether a biological sample has been exposed to both formaldehyde and acrolein. This study provides technical support for the rapid analysis of environmental exposure to acrolein and formaldehyde, as well as acrolein and formaldehyde modifications in proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the HPLC contrast chromatogram of the new adducts produced by the interaction of formaldehyde, acrolein and N-acetyl lysine;

[0025] Figure 2 This is the ultraviolet absorption spectrum of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared by the present invention;

[0026] Figure 3 This is a high-resolution mass spectrum of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared in the present invention;

[0027] Figure 4 This is the primary mass spectrum (negative ion mode) of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared in the present invention;

[0028] Figure 5 The secondary mass spectrum (negative ion mode) and main fragment ion structures of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared by the present invention;

[0029] Figure 6 The purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention 1 H NMR spectrum;

[0030] Figure 7 The purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention 13 C NMR spectrum;

[0031] Figure 8 The purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention 13 C and DEPT-135NRM NMR spectra;

[0032] Figure 9 HSQC-NMR spectrum of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared in the present invention;

[0033] Figure 10 This is the HMBC-NMR spectrum of the purified formaldehyde-acrolein-N-acetyllysine adduct prepared by the present invention;

[0034] Figure 11 is the structural formula of the compound FMD-N-acetyl lysine;

[0035] Figure 12 Schematic diagram of HMBC correlation of compound FMD-N-acetyl lysine;

[0036] Figure 13 This is a SDS-PAGE comparison of bovine serum albumin modified with formaldehyde and acrolein and native bovine serum albumin;

[0037] Figure 14 To identify the distribution of novel modification sites in the structure of bovine serum albumin;

[0038] Figure 15 The MS / MS spectrum of the representative peptide of VTK (+106.04 Da) CCTESLVNR; b-fragment (blue peak) represents the N-terminal fragment, and y-fragment (red peak) represents the C-terminal fragment;

[0039] Figure 16 This is the MS / MS spectrum of the representative ASQSVSSNLAWYQQK (+106.04Da)PGQAPR peptide in plasma samples;

[0040] Figure 17 This is the distribution map of modification sites identified by mass spectrometry in various tissues in mice;

[0041] Figure 18 This is the MS / MS spectrum of the representative peptide fragment of VHLTDAEK (+106.04Da)AAVSGLWGK in mouse aorta. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The present invention constructs an adduct of formaldehyde, acrolein and N-acetyl lysine, the molecular formula of which is: 14 H 22 N2O4, molecular weight: 294.164, structural formula:

[0048]

[0049] Analysis revealed that formaldehyde and acrolein co-bind to the side chain amino group of lysine, resulting in an additional structure of 106.04 Da. This 106.04 Da increase can be used to determine whether a biological sample has been exposed to both formaldehyde and acrolein. Details are as follows:

[0050] Example 1 Preparation of formaldehyde-acrolein-N-acetyllysine adduct

[0051] 1.5 mL of phosphate-buffered saline (PBS, pH 7.4, 20 mM) containing FA (20 mM), ACR (20 mM), and N-acetyl lysine (60 mM) was reacted at 37°C for 2 hours; 1.5 mL of phosphate-buffered saline (PBS, pH 7.4, 20 mM) containing FA (20 mM) and N-acetyl lysine (60 mM) was reacted at 37°C for 2 hours; and 1.5 mL of phosphate-buffered saline (PBS, pH 7.4, 20 mM) containing ACR (20 mM) and N-acetyl lysine (60 mM) was reacted at 37°C for 2 hours. The resulting products were then analyzed using an HPLC system (Shimadzu, Kyoto, Japan). For analysis, 3 μL of the membrane-filtered sample was injected and separated on a Zorbax SB-Aq column (4.6 × 250 mm, 5 μm) with a mobile phase of 0.1% formic acid / methanol (95:5). The column temperature was set to 40°C and the flow rate was maintained at 0.6 mL / min. The detection wavelength was 266 nm. The reaction product was detected by full wavelength scanning at a wavelength of 266 nm by high performance liquid chromatography (HPLC). According to the results of high performance liquid chromatography detection and nuclear magnetic resonance spectrum, the purity of the adduct prepared by the method of the present invention is as high as 99.6%, wherein the peak purity of the target single peak at each display wavelength (200-400 nm) of the high performance liquid chromatogram is as high as 99.6%. N-acetyl lysine reacts with FA and ACR to form a new compound (retention time of the target compound Rt = 11.1 minutes), which is different from the product formed by each aldehyde alone ( Figure 1 ).

[0052] In order to remove the residual aldehyde, the reaction solution was evaporated in vacuo at 45°C. Subsequently, 1.0 mL of water was added to dissolve the sample. The dissolved sample was then separated and purified using a chromatographic column (30 mm × 80 cm) filled with 350 mL of ODS C18 reverse phase silica gel. The fractions containing the desired product were lyophilized and analyzed by tandem mass spectrometry (MS / MS), high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). High resolution mass spectrometry analysis of the adduct of N-acetyl lysine with FA and ACR showed that its molecular weight was 294.164 Da and the molecular formula was C 15 H 22 N2O4( Figure 3 This molecular weight corresponds to the sum of N-acetyl lysine (188 Da), two molecules of ACR (56 Da), and one molecule of FA (30 Da), minus the dehydration effect of two water molecules: 188 + 2 × 56 + 30 - 2 × 18 = 294 Da. The proposed structure of the adduct is shown in Figure 5 The structure was consistent with the MS / MS data and was further confirmed by nuclear magnetic resonance (NMR) analysis (Table 1).

[0053] NMR spectroscopy ( Figures 6-10 ) showed a distinct aldehyde proton peak at δH 9.52 (1H, s, H-1). In the olefin region, significant signals appeared at δH 7.53 (1H, s, H-3), 5.93 (1H, s, H-7a), and 5.80 (1H, s, H-7b). The aliphatic region showed overlapping aminomethyl proton signals at δH 4.17 (5H, overlapping, H-5, H-6, H-12), and methylene proton signals at δH 3.29 (2H, m, H-8), 1.83 (2H, m, H-11), 1.72 (2H, m, H-9), and 1.43 (2H, m, H-10). A methyl resonance was also observed at δH 2.03 (3H, s, H-15).

[0054] 13 C NMR ( Figure 7 ) An aldehyde carbon was found at δC 193.46 (C-14), and ketone carbons were found at δC 178.72 (C-14) and 173.56 (C-15). Olefin carbons were observed at δC 144.90 (C-3), 132.31 (C-4), 131.40 (C-2), and 127.58 (C-7), while aminomethyl carbons were found at δC 55.51 (C-8), 54.62 (C-12), 53.20 (C-5), and 47.09 (C-6). Aliphatic carbons were identified at δC 30.89 (C-11), 23.20 (C-9), 22.16 (C-10), and 21.85 (C-15).

[0055] HMBC correlation ( Figure 10 ) showed that H-1 correlated with C-2 and C-6, H-3 correlated with C-1, C-7, C-5, and C-6, and the olefin proton H-7 correlated with C-3, C-4, and C-5, indicating that this was a pyridine aldehyde fragment. Further correlations, including H-15 with C-14, H-12 with C-13, C-14, and C-10, and H-9 and H-11 with C-10, supported the structure of the amide chain hexanoic acid fragment. The correlation of H-8 with C-9, C-5, and C-6 confirmed that the two fragments were connected via C-8 and nitrogen. Together, these data confirmed the structure of (S)-2-acetamido-6-(5-formyl-3-methylene-2,6-dihydropyridin-1(2H)-yl)hexanoic acid.

[0056] Table 1 NMR data of adducts of N-acetyl lysine interacting with acrolein and formaldehyde (D2O as solvent)

[0057]

[0058] Based on these findings, the reaction product of formaldehyde, acrolein, and N-acetyl-lysine was identified as a novel FA-ACR-N-acetyl-lysine adduct, referred to herein as FMD-lysine (N-Lys-FA-ACR). Structural analysis revealed that formaldehyde and acrolein co-bind to the side chain amino group of lysine, forming a 106.04 Da structural addition.

[0059] Example 2 Detection of modification sites in bovine serum albumin modified with formaldehyde acrolein

[0060] The bovine serum albumin sequence targeted in this example is PDB ID: 4F5S_B in NCBI (http: / / www.ncbi.nlm.nih.gov).

[0061] FA and ACR co-react with lysine residues to form a new adduct - 5-aldehyde-3-methylene-2,6-dihydropyridine-lysine (FMD-lysine), which increases the molecular weight of the lysine side chain in BSA by 106.04 Da. This modification may lead to a decrease in the positive charge of the protein. Based on these findings, the present invention speculates that the formation of FMD adducts is shown in Figure 13 In the strip shown by the red rectangle.

[0062] To confirm this, the peptide was digested in gel. BSA (38 mg / mL) was mixed with FA and ACR (5.80 mM) in 1.5 mL of PBS (pH 7.4, 20 mM). The mixture was then incubated at 37°C for 24 hours. For SDS-PAGE analysis, each sample treated with 5.80 mM aldehyde was subjected to SDS-PAGE. After electrophoresis, the gel was digested in gel to facilitate mass spectrometry analysis of the FA-ACR modifications.

[0063] For SDS-PAGE analysis, 20 μL of modified and unmodified BSA solutions (at a concentration of 1.25 μg / μL) were loaded onto the plate along with the markers. Electrophoresis was performed in a Tetra vertical electrophoresis tank at a constant voltage of 100 V for 90 minutes. Protein bands were then stained with Coomassie Brilliant Blue Rapid Stain and shaken at 40 rpm for 20 minutes. The gel was then stripped with Coomassie Brilliant Blue Stripper for 1 hour. Images of the stripped gel were captured using ImageJ software.

[0064] Then, perform in-gel digestion. Excise the protein bands obtained by SDS-PAGE and cut into 1 mm² gel pieces. Rinse the gel pieces three times with ultrapure water. Destain with a solution containing 50% acetonitrile (ACN) and 50 mM ammonium bicarbonate (NH4HCO3) at 37°C and 140 rpm for 1 hour. After thoroughly rinsing with ultrapure water, immerse the gel pieces in 100% acetonitrile until they turn white and harden. Then, dry them at 37°C.

[0065] The gel pieces were placed in a mixture of 50 mM NH4HCO3 and 10 mM DTT and incubated at 56°C for 1 hour to reduce them. For alkylation, the gel pieces were treated with a solution of 50 mM NH4HCO3 and 50 mM iodoacetamide for 30 minutes at room temperature in the dark. The gel pieces were then soaked in 100% ACN until they turned white and hardened again, and then dried.

[0066] Trypsin was added to the gel fragments in 50 mM NH₄HCO₃ and incubated at 37°C for 16 hours to promote digestion. After the incubation period, the supernatant was collected and the gel fragments were extracted with a 67% ACN and 2% formic acid solution at 37°C for 30 minutes. The gel fragments were then sonicated for 15 minutes and centrifuged. The resulting supernatant was mixed with the extract and freeze-dried.

[0067] For mass spectrometry analysis, lyophilized samples were reconstituted in 0.1% formic acid solution. The target concentration of samples for DDA mode analysis was 0.2 μg / μL. An Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) was used to collect MS spectra in DDA mode. The raw DDA data were processed and analyzed using MaxQuant version 2.1.4.0 (Max Planck Institute). The search parameters included methylation (C) as a fixed modification and oxidation (M) as a variable modification, protein N-terminal acetylation, and FMD modification of lysine (K, +106.04 Da). Trypsin / P was selected for enzyme digestion. The initial search mass tolerance was set to 20 ppm, and the main search mass tolerance was set to 4.5 ppm. In addition, a secondary mass spectrometry matching tolerance of 20 ppm was also adopted. The screening criteria at the protein and peptide levels were a false discovery rate (FDR) of 1%.

[0068] MaxQuant analysis identified 33 potential FMD modification sites in the hydrolyzed peptides (Table 2, Figure 14 Since MaxQuant can only report the total molecular weight of peptides, MS / MS was used to further verify the specific modification sites. In the MS / MS spectrum, peptide bonds are randomly cleaved during bombardment, producing smaller fragments, which help to accurately locate the modification sites. The most commonly observed fragments are y and b fragments, which are generated by cleavage at the N-terminus and C-terminus of the peptide chain, respectively. For example, in the peptide VTKCCTESLVNR( Figure 15 ), fragment b3 corresponds to the sequence VTK, while fragment b2 corresponds to VT. The mass difference between fragments b3 and b2 (435-201 = 234 Da) indicates an increase in the molecular weight of the lysine residue by 106.04 Da. This difference is calculated by subtracting the 18 Da loss due to dehydration from the molecular weight of the lysine residue (146 Da) (146-18 = 128 Da). Therefore, the observed difference of 234 Da corresponds to 106.04 Da. This increase of 106.04 Da is consistent with the observed mass shift from N-acetyllysine to FMD-N-acetyllysine. Therefore, these data confirm the presence of FMD modification on the remaining lysine residues in the protein.

[0069] Table 2 Identified FMD-lysine modified BSA peptides

[0070]

[0071]

[0072] Example 3 Detection of modification sites in plasma proteins

[0073] Four plasma samples obtained from human volunteers at risk for acrolein and formaldehyde exposure (HbA1c levels of 12.3%, 10.8%, 15.5%, and 12.1%, respectively) were precipitated with acetone. The precipitated proteins were reconstituted in PBS (pH 7.4, 20 mM) containing 8 M urea and adjusted to a concentration of 1 mg / mL. The protein solution was then treated with DTT and IAA. Urea was then replaced with NH4HCO3, and the protein concentration was adjusted to 2 mg / mL. Trypsin was then added at a 1:50 ratio to hydrolyze the protein. After 16 hours of digestion, 0.4% trans-fatty acids were added to inactivate the trypsin. The digests were separated using 4.0 mL ultrafiltration tubes (3 kDa molecular weight cutoff) and centrifuged at 7,500 × g for 40 minutes. The hydrolyzed peptides were collected in the ultrafiltration permeate. The hydrolyzed peptide solution was desalted using a CNWBOND LC-C18 SPE cartridge. The collected samples were then freeze-dried and dissolved in 0.1% formic acid solution to a peptide concentration of 1.0 mg / mL. iRT-Standard was added to each sample at a ratio of 10% of the total peptide concentration for data-independent acquisition (DIA) mode mass spectrometry analysis. DIA raw data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Modification identification of DIA raw data was performed using Spectronaut software (Omicsolution Co., Ltd., Shanghai, China). The search parameters included aminomethylation (C) as a fixed modification and FMD (K, +106.04 Da) as a variable modification. The false discovery rate (FDR) of protein identification was 1%. The analysis was performed based on the UniProt human protein database (http: / / www.uniprot.org). Mass spectrometry was used for analysis. The results (Table 3) showed that FMD-lysine was present in the blood samples (site probability greater than 0.75), which was further confirmed by the y fragment and b fragment information in the MS / MS spectrum ( Figure 16 ).

[0074] Table 3 Results of detecting modification sites in plasma proteins

[0075]

[0076]

[0077]

[0078] Example 4 Detection of modification sites in aldehyde-exposed mice

[0079] Six-week-old male C57BL / 6 mice were housed in a temperature- and humidity-controlled room with a 12-hour light / 12-hour dark cycle. Throughout the study, mice had free access to filtered water and standard mouse chow. After a one-week acclimation period in the animal facility, they were divided into two groups: six mice were exposed to FA and ACR, while a control group of six mice was exposed to air. The experimental group of mice was exposed to FA and ACR for 8 hours per day for 5 days in an 18-liter sealed transparent box. A nebulizer atomized FA and ACR solutions (distilled water was atomized for the control group) for one minute to achieve FA and ACR concentrations of 3.0 ppm in the enclosed environment. At the end of each hour, the box lid was opened and the aerosolized aldehydes were replaced with fresh air. After the box lid was replaced, the aldehyde solution was atomized again to maintain a consistent FA and ACR concentration of 3.0 ppm at the beginning of each hour. The exposure apparatus was placed in a fume hood throughout the experiment.

[0080] At the end of the exposure period, the mice were anesthetized and blood samples were collected. The animals were then euthanized, and tissues, including the heart, liver, lungs, kidneys, hippocampus, aorta, remaining brain tissue (excluding the hippocampus), and plasma, were collected. These tissues were snap-frozen in liquid nitrogen and stored at -80°C until further analysis.

[0081] Mouse tissue protein was extracted using a Seven Fast Column Total Protein Extraction Kit. Protein was precipitated with acetone. The precipitated protein was reconstituted in PBS (pH 7.4, 20 mM) containing 8 M urea and adjusted to a concentration of 1 mg / mL. The protein solution was then treated with DTT and IAA. Urea was then replaced with NH₄HCO₃, and the protein concentration was adjusted to 2 mg / mL. Trypsin was then added at a ratio of 1:50 to hydrolyze the protein. After 16 hours of digestion, the trypsin solution was inactivated by the addition of 0.4% trifluoroacetic acid. The enzymatic product was separated using a 4.0 mL ultrafiltration tube (3 kDa molecular weight cutoff) and centrifuged at 7,500 × g for 40 minutes. The hydrolyzed peptides were collected in the ultrafiltration permeate. The hydrolyzed peptide solution was desalted using a CNWBOND LC-C18 SPE cartridge. The collected sample was then lyophilized and dissolved in 0.1% formic acid to a peptide concentration of 1.0 mg / mL. iRT-Standard was added to each sample at a ratio of 10% of the total peptide concentration for data independent acquisition (DIA) mode mass spectrometry analysis. DIA raw data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Modification identification was performed on the DIA raw data using Spectronaut software (Omicsolution Co., Ltd., Shanghai, China). Search parameters included aminomethylation (C) as a fixed modification and FMD (K, +106.04 Da) as a variable modification. The false discovery rate (FDR) for protein identification was 1%. Analysis was performed according to the UniProt mouse protein database (http: / / www.uniprot.org).

[0082] Data independent acquisition (DIA) analysis (site probability greater than 0.75) showed that a total of 231 FMD modification sites were identified in the heart, liver, lung, kidney, hippocampus, brain (excluding hippocampus), and aorta (Table 4). The hippocampus had the most modification sites (38), while plasma had the fewest modification sites ( Figure 17 The y and b fragment information in the MS / MS spectrum confirmed this modification ( Figure 18 The widespread detection of FMD modifications in different organs and tissues suggests that FMD may serve as a novel biomarker for assessing co-exposure to FA and ACR.

[0083] Table 4 Results of detecting modification sites in aldehyde-exposed mice

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting acrolein and formaldehyde exposure, characterized in that The following steps are involved: Perform protein reconstruction and enzymatic hydrolysis on the protein sample to be tested to obtain an enzymatic hydrolyzate; Performing mass spectrometry analysis on the enzymatic hydrolysate; when a lysine residue shows a molecular weight increase of 106.04 Da, it is determined that the protein sample to be tested has been exposed to acrolein and formaldehyde simultaneously; placing the protein sample to be tested in a PBS solution containing urea to perform protein reconstitution; The protease used in the enzymatic hydrolysis treatment is trypsin.

2. The method according to claim 1, characterized in that The concentration of urea in the PBS solution was 8 M.

3. A method for identifying chemically modified sites of lysine in acrolein and formaldehyde, characterized in that: The following steps are involved: Perform protein reconstruction and enzymatic hydrolysis on the protein sample to be tested to obtain an enzymatic hydrolyzate; Performing mass spectrometry analysis on the enzymatic hydrolysate, when a molecular weight increase of 106.04 Da is observed at the lysine residue site, the lysine residue site is determined to be a lysine chemical modification site of acrolein and formaldehyde; placing the protein sample to be tested in a PBS solution containing urea to perform protein reconstitution; The protease used in the enzymatic hydrolysis treatment is trypsin.

4. The method according to claim 3, characterized in that The concentration of urea in the PBS solution was 8 M.

5. An adduct of formaldehyde, acrolein and N-acetyl lysine, characterized in that: The structural formula is as follows: 。 6. Use of the adduct according to claim 5 in detecting exposure to acrolein and formaldehyde.

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