Method for detecting acrolein and formaldehyde exposure

Abstract

The invention discloses a method for detecting acraldehyde and formaldehyde exposure, and relates to the technical field of biology. The method comprises the following steps: performing protein reconstruction and enzymolysis treatment on a to-be-detected protein sample to obtain enzymatic hydrolysate; and carrying out mass spectrometry on the enzymatic hydrolysate, and when the molecular weight of the lysine residue is increased by 106.04 Da, judging that the to-be-detected sample is simultaneously exposed to acrolein and formaldehyde environments. Through construction of an adduct of formaldehyde, acrolein and N-acetyl lysine, analysis finds that the molecular structure of 106.04 Da can be increased when formaldehyde and acrolein are jointly combined to side chain amino of lysine. The molecular structure of 106.04 Da of a protein lysine side chain is increased, so that whether a biological sample is simultaneously exposed to formaldehyde and acrolein environments or not can be judged. Technical support is provided for rapid analysis of environmental exposure of acrolein and formaldehyde and modification of acrolein and formaldehyde in protein.

Description

Technical Field

[0001] The 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), which are derived from both endogenous and exogenous sources. Moreover, FA and ACR are widely present in food, and human exposure is high in daily life. RCSs are extremely reactive in nature, attacking and modifying biomacromolecules such as proteins, DNA, and RNA in the body, which is harmful to human health. As for ACR, four ACR-induced protein adducts have been found: Schiff base of lysine, Michael adducts of cysteine, histidine and lysine, N-(3-formyl-3,4-dehydropiperidinyl lysine (FDP-lysine) and N-ε-3-methylpyridinyl lysine (MP-lysine). On the other hand, formaldehyde usually cross-links proteins through a methylene bridge mechanism. Specifically, it forms Mannich bases with free amino groups (such as lysine residues), and then undergoes chemical modification through interactions with other amino groups, resulting in intermolecular and intramolecular 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), and its endogenous conversion rate is as high as 878-1310 mg / kg per day. Acrolein has been listed as a high-priority hazardous air pollutant by relevant units. The formation of acrolein and formaldehyde in the environment, food and body is simultaneous, and the two harmful aldehydes often coexist in the above exposure environments, but there is a lack of research on the structure and site of proteins co-modified by acrolein and formaldehyde, and there is a lack of methods for detecting co-exposure of acrolein and formaldehyde.

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

[0004] The purpose of the present invention is to provide a method for detecting acrolein and formaldehyde exposure to solve the problems of the above-mentioned prior art. The present invention provides technical support for the rapid analysis of environmental exposure to acrolein and formaldehyde and acrolein and formaldehyde modification 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 to 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, and when a molecular weight increase of 106.04 Da appears 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 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 the use of the above adduct in detecting acrolein and formaldehyde exposure.

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

[0022] The present invention constructs an adduct of formaldehyde, acrolein and N-acetyl lysine, and analyzes and finds that when formaldehyde and acrolein are combined with the side chain amino group of lysine, a molecular structure of 106.04 Da will be increased. By using the increase of the molecular structure of 106.04 Da on the side chain of protein lysine, it can be judged whether the biological sample is exposed to formaldehyde and acrolein 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 modification 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 drawings required for use in the embodiments will be briefly introduced below. 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 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 The ultraviolet absorption spectrum of the purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention;

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

[0027] Figure 4 The primary mass spectrum (negative ion mode) of the purified product of formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention;

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

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

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

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

[0032] Fig. 9 HSQC-NMR spectrum of the purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention;

[0033] Fig.10 The HMBC-NMR spectrum of the purified formaldehyde-acrolein-N-acetyl lysine adduct prepared by the present invention;

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

[0035] Fig.12 It is a schematic diagram of HMBC correlation of compound FMD-N-acetyl lysine;

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

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

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

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

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

[0041] Fig.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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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 found that formaldehyde and acrolein combined with the side chain amino group of lysine will increase the structure of 106.04Da. The increase of 106.04Da structure can be used to determine whether the biological sample is exposed to formaldehyde and acrolein at the same time. The details are as follows:

[0050] Example 1 Preparation of formaldehyde-acrolein-N-acetyl-lysine 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; 1.5 mL of phosphate buffered saline (PBS, pH7.4, 20 mM) containing ACR (20 mM) and N-acetyl lysine (60 mM) was reacted at 37 ° C for 2 hours. The generated products were then analyzed using an HPLC system (Shimadzu, Kyoto, Japan). During the 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 HPLC 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 HPLC 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. Then, the dissolved sample was 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 is equivalent 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 As shown, the structure was consistent with the MS / MS data and was further confirmed by nuclear magnetic resonance (NMR) analysis (Table 1).

[0053] NMR spectroscopy ( Figure 6-Figure 10 ) showed an obvious 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, overlap, 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). 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 ( Fig.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 determined to be a new type of FA-ACR-N-acetyl lysine adduct, referred to as FMD-lysine (N-Lys-FA-ACR) in the present invention. Through structural analysis, it can be seen that formaldehyde and acrolein will be combined with the side chain amino group of lysine to form a structural increase of 106.04Da.

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

[0060] The bovine serum albumin sequence targeted by 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 Fig.13 In the strip shown by the red rectangle.

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

[0063] For SDS-PAGE analysis, 20 μL of modified and unmodified BSA solution (concentration of 1.25 μg / μL) were loaded into Tetra vertical electrophoresis tank. Electrophoresis was performed at a constant voltage of 100 V for 90 minutes. Subsequently, protein bands were stained with Coomassie Brilliant Blue Fast Staining Solution and shaken at 40 rpm for 20 minutes. The gel was then destained with Coomassie Brilliant Blue Destaining Solution for 1 hour. Images of the destained 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 blocks. Then rinse the gel blocks three times with ultrapure water. Destain at 37°C and 140rpm for 1 hour using a solution containing 50% acetonitrile (ACN) and 50mM ammonium bicarbonate (NH4HCO3). After thorough washing with ultrapure water, immerse the gel blocks in 100% acetonitrile until they turn white and hard. Then dry 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, the supernatant was collected and the gel fragments were extracted with 67% ACN and 2% formic acid solution at 37°C for 30 minutes. The gel fragments were then ultrasonicated 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. Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) was used to collect MS spectra in DDA mode. The original DDA data were processed and analyzed using MaxQuant 2.1.4.0 version (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, +106.04Da). Trypsin / P was selected for enzyme digestion. The initial search mass tolerance was set to 20ppm, and the main search mass tolerance was set to 4.5ppm. In addition, a secondary mass spectrometry matching tolerance of 20ppm was used. 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, Fig.14 ). Since MaxQuant can only report the total molecular weight of a peptide, specific modification sites were further verified using MS / MS. In MS / MS spectra, peptide bonds are randomly cleaved upon bombardment, producing smaller fragments that help pinpoint the modification site. The most commonly observed fragments are the y and b fragments, which result from cleavage at the N-terminus and C-terminus of the peptide chain, respectively. For example, in the peptide VTKCCTESLVNR( Fig.15 ), the b3 fragment corresponds to the sequence VTK, while the b2 fragment corresponds to VT. The mass difference between the b3 and b2 fragments (435-201=234Da) indicates that the molecular weight of the lysine residue has increased by 106.04Da. The calculation method is to subtract the 18Da lost by dehydration (146-18=128Da) from the molecular weight of the lysine residue (146Da), so the observed difference of 234Da is equivalent to 106.04Da. The increase of 106.04Da is completely consistent with the mass transfer observed from N-acetyllysine to FMD-N-acetyllysine. Therefore, these data confirm the presence of FMD modification on the residual lysine 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 of acrolein and formaldehyde exposure (HbA1c levels of 12.3%, 10.8%, 15.5%, and 12.1%, respectively) were selected and proteins were precipitated with acetone. The precipitated proteins were reconstituted in PBS (pH 7.4, 20mM) containing 8M urea and the concentration was adjusted to 1mg / mL. The protein solution was then treated with DTT and IAA. After that, urea was replaced with NH4HCO3, and the protein concentration was adjusted to 2mg / mL, and trypsin was added at a ratio of 1:50 to hydrolyze the protein. After 16 hours of digestion, 0.4% trans fatty acids were added to inactivate trypsin. The enzymatic products were separated using a 4.0mL ultrafiltration tube (3kDa molecular 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 lyophilized 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 ( Fig.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-h light / 12-h dark cycle per day. Mice had free access to filtered water and standard mouse chow throughout the study. After a one-week acclimation period in the animal facility, mice were divided into two groups, six mice were exposed to FA and ACR, while a control group consisting of six mice were exposed to air. The experimental group mice were exposed to FA and ACR for 8 h per day for 5 days, and the mice were placed in an 18-L sealed transparent box for exposure. The nebulizer atomized the FA and ACR solutions (the control group atomized distilled water) for one minute, thereby achieving a FA and ACR concentration of 3.0 ppm in the closed environment. At the end of each hour, the box lid was opened to replace the aerosolized aldehydes 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, mice were anesthetized and blood samples were collected. The animals were then euthanized and tissues, including heart, liver, lung, kidney, hippocampus, aorta, remaining brain tissue (excluding hippocampus), and plasma, were collected. These tissues were snap-frozen in liquid nitrogen and stored at -80°C for further analysis.

[0081] Mouse tissue protein was extracted using the 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 the concentration was adjusted to 1 mg / mL. The protein solution was then treated with DTT and IAA. After that, urea was replaced with NH4HCO3, and the protein concentration was adjusted to 2 mg / mL, and trypsin was added at a ratio of 1:50 to hydrolyze the protein. After 16 hours of digestion, 0.4% trifluoroacetic acid was added to inactivate the trypsin solution. The enzymatic hydrolyzate was separated using a 4.0 mL ultrafiltration tube (3 kDa molecular 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 lyophilized and dissolved in a 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). Spectronaut software (Omicsolution Co., Ltd., Shanghai, China) was used to identify modifications in DIA raw data. Search parameters included aminomethylation (C) as a fixed modification and FMD (K, +106.04Da) as a variable modification. The false discovery rate (FDR) for protein identification was 1%. The 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 tissue (excluding hippocampus) and aorta (Table 4). The hippocampus had the most modification sites (38), while plasma had the least modification sites ( Fig.17 The y and b fragment information in the MS / MS spectrum confirmed this modification ( Fig.18 ). The widespread detection of FMD modifications in different organs and tissues suggests that FMD could serve as a novel biomarker for assessing co-exposure to FA and ACR.

[0083] Table 4 Results of detecting modification sites in mice exposed to aldehydes

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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; The enzymatic hydrolysate is subjected to mass spectrometry analysis. When the molecular weight of the lysine residue increases to 106.04 Da, it is determined that the sample to be tested has been exposed to acrolein and formaldehyde environments at the same time.

2. The method according to claim 1, characterized in that: The protein sample to be tested is placed in a PBS solution containing urea for protein reconstitution.

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

4. The method according to claim 1, characterized in that: The protease used in the enzymatic treatment is trypsin.

5. A method for identifying chemical modification 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; The enzymatic hydrolysate is subjected to mass spectrometry analysis, and when a molecular weight increase of 106.04 Da appears at the lysine residue site, it is determined that the lysine residue site is a lysine chemical modification site of acrolein and formaldehyde.

6. The method according to claim 5, characterized in that The protein sample to be tested is placed in a PBS solution containing urea for protein reconstitution.

7. The method according to claim 6, characterized in that The concentration of the urea in the PBS solution was 8M.

8. The method according to claim 5, characterized in that The protease used in the enzymatic treatment is trypsin.

9. An adduct of formaldehyde, acrolein and N-acetyl lysine, characterized in that: The structural formula is as follows:

10. Use of the adduct according to claim 9 in detecting exposure to acrolein and formaldehyde.

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