TY - JOUR
AU1 - Sýkora,, David
AU2 - Jindřich,, Jindřich
AU3 - Král,, Vladimír
AU4 - Jakubek,, Milan
AU5 - Tatar,, Ameneh
AU6 - Kejík,, Zdeněk
AU7 - Martásek,, Pavel
AU8 - Zakharov,, Sergey
AB - Abstract Methanol, an aliphatic alcohol widely used in the industry, causes acute and chronic intoxications associated with severe long-term health damage, including permanent visual impairment, brain damage, mainly necrosis of the basal ganglia and high mortality due to cancer. However, the role of formaldehyde, an intermediate metabolite of methanol oxidation, in methanol toxicity remains unclear. Thus, we studied the reactivity of several amino acids and peptides in the presence of formaldehyde by identifying products by direct infusion electrospray high-resolution mass spectrometry (MS) and matrix-assisted laser desorption-ionization MS. Cysteine, homocysteine and two peptides, CG and CGAG, provided cyclic products with a +12 amu mass shift with respect to the original compounds. The proposed structures of the products were confirmed by high-resolution tandem MS. Moreover, the formation of the products with +12 amu mass shift was also shown for two biologically relevant peptides, fragments of ipilimumab, which is a human IgG1 monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4. Overall, our experimental results indicate that formaldehyde reacts with some amino acids and peptides, yielding covalently modified structures. Such chemical modifications may induce undesirable changes in the properties and function of vital biomolecules (e.g., hormones, enzymes) and consequently pathogenesis. Introduction Methanol is a toxic aliphatic alcohol widely applied in the industry, agriculture and cleaning services as a solvent, coolant, fuel and component of windshield washer and defrosting fluids and as a primary chemical for the synthesis of other compounds (1–3). Cases of accidental or professional exposure to methanol by inhalation or ingestion, which causes symptoms of acute, subacute or chronic intoxication, are reported worldwide (4–7). Mass or cluster methanol poisoning outbreaks due to consumption of illicit alcohol containing a high proportion of methanol remain a major public health problem associated with high mortality rates and severe long-term health impairment in survivors (8–10). Methanol is far more toxic than ethanol. As little as 10–15 mL of pure methanol can cause permanent blindness due to neuronal damage to the optic nerve, and 30 mL may be potentially fatal if treatment is delayed or inadequate (11). Methanol is metabolized to formaldehyde by alcohol dehydrogenase, mostly in the liver. Subsequently, formaldehyde is further converted by aldehyde dehydrogenase into formic acid, which inhibits cytochrome c oxidase in mitochondria, resulting in ATP depletion, lactate accumulation and development of severe metabolic acidosis with a high anion gap (12, 13). The mechanisms of methanol toxicity; however, may be related to the first methanol metabolite, formaldehyde, which is also highly reactive. Formaldehyde is hematotoxic to mice and humans (14) and has also been classified as a human leukemogen by the International Agency for Research on Cancer (15) and by the US National Toxicology Program (16). Recently, a study has shown that patients with amyotrophic lateral sclerosis have significantly higher plasma levels of formaldehyde than controls, thus suggesting that formaldehyde is neurotoxic and may contribute to pathogenesis (17). In addition, formaldehyde genotoxicity has been studied and confirmed as well (18). In BALB/c mice, formaldehyde has been shown to induce apoptosis in bone marrow cells (19). In humans subjected to acute methanol poisoning, 47% of those who died during the 6-year follow-up period, died due to different cancers: prostate, pancreatic, esophageal and lung cancer and acute leukemia (20). Yet, despite these findings, the exact mechanism underlying the pathological effects of formaldehyde remains unclear. Formaldehyde is a very reactive compound. Thus, direct chemical reactions with various biologically relevant compounds, amino acids (AAs), peptides and proteins, are widely used in bio-orthogonal chemical transformations (21). Among the reactions of formaldehyde with AAs published thus far, the reactions with the primary amino group of free AAs, with the amino group of the lysine side chain or with the N-terminal amino group of peptides or proteins, stand out (22). Under physiological conditions, the fastest reaction of formaldehyde with an amino group is the formation of a quaternary ammonium intermediate (R-N+H2CH2OH), which is dehydrated to iminium cation (R-N+H=CH2). This iminium cation can readily react with nucleophilic groups (NuH) such as amino, sulfanyl or phenolic hydroxyl groups, which are usually close in proteins or peptides; thus, formaldehyde behaves as a bifunctional linker by forming the R-NH-CH2-Nu conjugate. In addition to this reaction, common for all AAs, other specific reactions with some AAs have also been published. For tryptophan and histidine, the Pictet-Spengler reaction with aldehydes (23) yields a product with one additional heterocycle. In the case of formaldehyde, the reaction product contains a covalent bond between an aromatic carbon and the amino group through the methylene linker. In turn, cysteine and homocysteine react with aldehydes, forming thiazolidine or thiazinane derivatives, respectively; because this reaction is fast, it can be used to detect these AAs (24). Based on the above, we hypothesize that protein dysfunction can be induced via covalent modification by formaldehyde through the aforementioned reactions whereby various products can be formed. To test our hypothesis, we performed a series of proof-of-concept experiments toward establishing an experimental paradigm for the identification of products from reactions between formaldehyde and AAs. Experimental Chemicals and solvents The following AAs and peptides were studied: l-cysteine, d,l-homocysteine, l-lysine, glycinamide hydrochloride, l-methionine, l-serine (all Sigma-Merck, Czech Republic); CG and CGAG (both synthesized at the Institute of Organic Chemistry and Biochemistry, Czech Republic); and SSYTMHWVRQAPGKGLEWVTFISYDGNNKY (SY30) and VGSSYLAWYQQKPGQAPRLLIYGAFSRATG (VG30) (both PepMic, China). All solvents used for dissolution of AAs and peptides were of LC-MS grade, specifically, methanol and acetonitrile purchased from Sigma-Merck, Czech Republic, ultrapure water (18 MΩ cm) produced with the system PureLab Ultra (Elga, UK), phosphate buffer saline (PBS) consisting of 10 mmol/L Na2PO4.2H2O, 1.8 mmol/L KH2PO4, 137 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.5 (Sigma-Merck, Czech Republic), and formaldehyde solution (36.5–38% in water), which was purchased from Sigma-Merck (Czech Republic). Instrumentation For mass spectrometric (MS) analysis, an Impact II, which is a Q-TOF hybrid high-resolution (HR) MS equipped with an electrospray ion source, and an Autoflex Speed matrix-assisted laser dissociation/ionization (MALDI) TOF-TOF (both Bruker, Germany) were used. Matrix solution for MALDI experiments In total, 20 mg of 2,5-dihydroxybenzoic acid (Merck-Sigma, Czech Republic) was dissolved in 1 mL of a solution of 3:7 (v/v) acetonitrile:0.1% trifluoroacetic acid in water. Results Formaldehyde is a very reactive compound. When in contact with AAs, peptides and proteins, formaldehyde may form various products. For instance, we have proposed the formation of at least four reaction products for l-cysteine (Figure 1). Analogous products can be expected for some other AAs. Figure 1 Open in new tabDownload slide Proposed products (and their exact masses for the corresponding [M + H]+ ions) of the reaction of L-cysteine with formaldehyde. Figure 1 Open in new tabDownload slide Proposed products (and their exact masses for the corresponding [M + H]+ ions) of the reaction of L-cysteine with formaldehyde. We performed MS experiments to confirm the formation of some of our proposed products. A solution of l-cysteine (1 mmol/L) in PBS buffer was further diluted with 1:1 (v/v) methanol/water in a 1:9 volume ratio. The resulting sample was directly infused into the ES ion source of the HRMS Q-TOF instrument, recording an MS spectrum (Figure 2A). Then, an equimolar amount of formaldehyde dissolved in water was added to the 1 mmol/L solution of l-cysteine in PBS, and the vial with the reaction mixture was thermostated in a water bath at 37°C. Small fractions were detracted continuously at defined time intervals, and after a 1:9 (v/v) dilution with methanol/water (1:1, v/v), the solution was analyzed by direct infusion with ES+ HRMS Q-TOF. The resulting MS spectrum obtained in 10 min reaction time is shown in Figure 2B. Figure 2 Open in new tabDownload slide (A) ESI+ HRMS spectrum of L-cysteine before adding formaldehyde; (B) ESI+ HRMS spectrum of L-cysteine plus formaldehyde, after 10 min of contact time. Figure 2 Open in new tabDownload slide (A) ESI+ HRMS spectrum of L-cysteine before adding formaldehyde; (B) ESI+ HRMS spectrum of L-cysteine plus formaldehyde, after 10 min of contact time. The product with an elemental composition corresponding to the structures (a)/(b) in Figure 1 was formed within several minutes in a very high yield. Figure 3 depicts the progress of the conversion of the sodiated l-cysteine ion, [M + Na]+ (exact mass 144.0090, found 144.0098), into the product [M + 12 + Na]+ (exact mass 156.0090, found 156.0097). The yield of the product reached ~95% within 10 min (Figure 3). The mass difference corresponds exactly to a mass gain of one carbon atom. Figure 3 Open in new tabDownload slide Formation of the product (a)/(b) in the mixture of L-cysteine with formaldehyde. Figure 3 Open in new tabDownload slide Formation of the product (a)/(b) in the mixture of L-cysteine with formaldehyde. Then, d,l-homocysteine and dipeptide CG and tetrapeptide CGAG were mixed with formaldehyde in a similar experiment. The rate and degree of conversion of these reactants were similar to those of l-cysteine, i.e., the mass gain was also one carbon atom (+12 amu). Several other AAs were subjected to the reaction with formaldehyde under the same conditions, including l-methionine and glycine amide. In the case of l-methionine, the reaction was slower, and the conversion reached 0.33% in 2 h. For glycine amide, we determined 0.68% of product +12 amu in the same time period. Conversely, experiments with l-serine and l-lysine provided no evidence of the formation of +12 amu products. The progress of +12 amu product formation for the studied compounds is summarized in Table I. Table I Formation of +12 amu product Compound . Conversion to +12 amu product (at 37°C) . in PBS . in H2O . L-cysteine ~95% in 10 min ~95% in 12 h D,L-homocysteine CG CGAG L-methionin 0.33% in 2 h no product found in 2 h glycine amide 0.68% in 2 h 0.45% in 2 h L-serine no product found no product found L-lysine in 24 h in 24 h Compound . Conversion to +12 amu product (at 37°C) . in PBS . in H2O . L-cysteine ~95% in 10 min ~95% in 12 h D,L-homocysteine CG CGAG L-methionin 0.33% in 2 h no product found in 2 h glycine amide 0.68% in 2 h 0.45% in 2 h L-serine no product found no product found L-lysine in 24 h in 24 h Open in new tab Table I Formation of +12 amu product Compound . Conversion to +12 amu product (at 37°C) . in PBS . in H2O . L-cysteine ~95% in 10 min ~95% in 12 h D,L-homocysteine CG CGAG L-methionin 0.33% in 2 h no product found in 2 h glycine amide 0.68% in 2 h 0.45% in 2 h L-serine no product found no product found L-lysine in 24 h in 24 h Compound . Conversion to +12 amu product (at 37°C) . in PBS . in H2O . L-cysteine ~95% in 10 min ~95% in 12 h D,L-homocysteine CG CGAG L-methionin 0.33% in 2 h no product found in 2 h glycine amide 0.68% in 2 h 0.45% in 2 h L-serine no product found no product found L-lysine in 24 h in 24 h Open in new tab In all our experiments, we also tried to find the hypothetical structures (c) and (d) proposed in Figure 1. However, their formation was inconclusive. In contrast, the formation of product +12 amu was evident, and conversion was also practically complete for l-cysteine, d,l-homocysteine, dipeptide CG and tetrapeptide CGAG in plain water. The experiments repeated under similar conditions, albeit replacing PBS by water, provided the expected [M + 12 + H]+ and [M + 12 + Na]+ ions. However, the reaction rate was significantly slower, e.g., for l-cysteine, 95% conversion into +12 amu product required ~12 h (Table I). Finally, we studied two peptides, SY30 and VG30, both consisting of 30 AAs. These two peptides are highly biologically relevant because they comprise constituents of ipilimumab, a human IgG1 monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab activates the immune system and is used to treat melanoma. Moreover, ipilimumab is currently undergoing clinical trials for the treatment of other cancers (25–27). Their analysis was based on both direct infusion of the samples into ES+ HRMS Q-TOF (data not shown) and MALDI measurements. For MALDI experiments: (a) the peptide SY30 was dissolved in water at a concentration of 100 μg/mL. The solution was further diluted in water to a final concentration of 2 μg/mL, and 2 μL of this solution was mixed with 2 μL of 2,5-DHB solution. Approximately 0.5 μL of the resulting mixture was placed on the MALDI target and left to evaporate to dryness. Lastly, the MALDI MS spectrum of the peptide was measured; (b) to 10 mL of the aqueous solution of SY30 (100 μg/mL), 10 μL of the formaldehyde solution (10× diluted with H2O) was added, and the vial was placed in a water bath thermostated at 37°C. After 48 h, 10 μL of the solution was collected and mixed with 490 μL of water to decrease the concentration to 2 μg/mL. Then, 2 μL of this solution was mixed with 2 μL of 2,5-DHB solution. Approximately 0.5 μL of the resulting mixture was placed on the MALDI target and left to evaporate to dryness. Lastly, the MALDI MS spectrum was measured. Figure 4 shows a representative MALDI spectrum of SY30 before adding formaldehyde versus a spectrum recorded after 48 h of contact with the formaldehyde solution, clearly showing the formation of the product with molecular mass + 12 amu. Figure 4 Open in new tabDownload slide MALDI spectra of SY30, MS spectrum in the absence of formaldehyde (magenta), and spectrum of SY30 in the presence of formaldehyde (blue) for 48 h. Figure 4 Open in new tabDownload slide MALDI spectra of SY30, MS spectrum in the absence of formaldehyde (magenta), and spectrum of SY30 in the presence of formaldehyde (blue) for 48 h. Similar results were found when testing the peptide VG30 (data not shown). The reaction rates of SY30 and VG30 were lower than those of l-cysteine, l-homocysteine, CG and CGAG, but the +12 amu products were identified in all cases. Discussion ESI with direct infusion enabled an easy and reliable tracking of formaldehyde reaction with AAs and peptides. The HR of the Impact II Q-TOF ESI instrument provides ionized products with traceable elemental compositions. Thus, conclusions related to the chemical composition of the formed ions are highly reliable. We used HRMS ESI not only for the study of products of the formaldehyde reaction with the AAs and peptides (for unambiguous confirmation of the formation of each product with a mass exactly corresponding to the mass gain relevant to one carbon atom) but also in subsequent experiments focused on the fragmentation of the +12 amu products. Thus, Figure 5A shows the product ion spectrum of [l-cysteine+12 + H]+ ion, wherein the proposed fragment ion, [C3H6NS]+ (exact mass 88.0215, found 88.0219), dominated the spectrum. Figure 5B depicts the spectrum of [d,l-homocysteine+12 + H]+ with the most intense proposed fragment ion, [C4H8NS]+ (exact mass 102.0372, found 102.0375). The second most intense fragment corresponds to [C3H6NO2]+ (exact mass 88.0397, found 88.0397). In both cases, the fragmentation of the relevant precursor led to the loss of formic acid. The fragments found in the product ion scans of both l-cysteine and d,l-homocysteine are in good agreement with the formaldehyde-induced formation of the cyclic structure (b) proposed in Figure 1. Figure 5 Open in new tabDownload slide Product ion ES+ MS spectra of (A) modified L-cysteine, [M + 12 + H]+ ion (m/z 134), and (B) modified D,L-homocysteine [M + 12 + H]+ (m/z 148) ion and the most intense fragments, at 20 eV collision energy. Figure 5 Open in new tabDownload slide Product ion ES+ MS spectra of (A) modified L-cysteine, [M + 12 + H]+ ion (m/z 134), and (B) modified D,L-homocysteine [M + 12 + H]+ (m/z 148) ion and the most intense fragments, at 20 eV collision energy. The reactivity of the peptides SY30 and VG30, which have molecular masses well above 3 kDa, was studied using the same approach as the AAs and short peptides, that is, HRMS ESI but this time also MALDI was used. Both techniques are highly suitable for peptide characterization, and they are frequently used together in the field of proteomics, complementing each other. MALDI spectra of peptides are simpler than the corresponding ESI MS spectra because, in the former, preferentially monoprotonated ions are formed during the ionization process. Conversely, peptides of moderate molecular mass, such as SY30 and VG30 provided multiply protonated and sodiated ions in ESI+ MS spectra, with most intense signals for five, four and three times charged precursor ions, thereby rendering the resulting spectrum MS more complex and less synoptic. We studied the mentioned peptides using both techniques. However, for the sake of lucidity, we present here only the MALDI results. Our specific MALDI instrumentation provided significantly lower mass resolution than the Q-TOF MS Impact II. Consequently, the measurement of the exact mass of the analytes was challenging, but the data gained by HRMS ESI+ clearly confirmed the formation of the product with exactly +12 amu mass in the reaction of the peptides SY30 and VG30. All aforementioned findings strongly support our hypothesis that formaldehyde modifies peptides/proteins at 37°C in both PBS (mimicking physiological conditions) and water. Conclusion The results indicate that formaldehyde reacts with AAs and peptides, forming covalent structures. Consequently, formaldehyde may modify the function of key enzymes, thereby causing pathogenicity. To further confirm our hypothesis, we are currently evaluating proteomic data on samples obtained from the Czech methanol mass poisoning outbreak, which occurred in September and December 2012, focusing on potential covalent protein modifications exposed to enhanced formaldehyde concentration. Disclosure statement The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper. The manuscript has been read and approved by all authors. The authors certify that the submission is not under review at any other publication. The authors certify that they have no other submissions and previous reports that might be regarded as overlapping with the current work. The authors declare no financial disclosures. Acknowledgment This work was supported by the Ministry of Health of the Czech Republic, the Project [16-27075A] of AZV VES 2016, and the Projects PROGRES [Q25] and [Q29], First Faculty of Medicine, Charles University in Prague, Czech Republic. We thank Miroslava Blechova (IOCB, Prague) for peptide synthesis. References 1. Blug , M. , Leker , J., Plass , L. Methanol: the basic chemical and energy feedstock of the future. In: Bertau M., Offermanns H., Plass L.F.S., Wernicke H.J. (eds). In Methanol generation economics . Springer : Berlin, Heidelberg , 2014 . 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( 2017 ) Ipilimumab: From preclinical development to future clinical perspectives in melanoma . Future Oncology , 13 , 625 – 636 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Deceased © The Author(s) 2020. Published by Oxford University Press on behalf of The International Association of Forensic Toxicologists, Inc. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Formaldehyde Reacts with Amino Acids and Peptides with a Potential Role in Acute Methanol Intoxication
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkaa039
DA - 2020-12-12
UR - https://www.deepdyve.com/lp/oxford-university-press/formaldehyde-reacts-with-amino-acids-and-peptides-with-a-potential-1HG8Rmhqy5
SP - 880
EP - 885
VL - 44
IS - 8
DP - DeepDyve
ER -