Identification of Formaldehyde-induced Modifications in Proteins
Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde. Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde. Aldehydes, such as formaldehyde and glutaraldehyde are widely employed reagents in the biochemical, biomedical, and pharmaceutical fields. Formaldehyde, for example, is applied to inactivate toxins and viruses for the production of vaccines, such as diphtheria, tetanus toxoid, hepatitis A, anthrax, and inactivated polio vaccine, and to stabilize recombinant pertussis toxin (1Rappuoli R. Levine M.M. Woodrow G.C. Kaper J.B. Cobon G.S. New Generation Vaccines. 2nd Ed. Marcel Dekker, Inc., New York1997: 417-435Google Scholar, 2Stapleton J.T. Lemon S.M. Levine M.M. Woodrow G.C. Kaper J.B. Cobon G.S. New Generation Vaccines. 2nd Ed. Marcel Dekker, Inc., New York1997: 417-435Google Scholar, 3Leppla S.H. Robbins J.B. Schneerson R. Shiloach J. J. Clin. Invest. 2002; 110: 141-144Crossref PubMed Scopus (118) Google Scholar, 4Murdin A.D. Barreto L. Plotkin S. Vaccine. 1996; 14: 735-746Crossref PubMed Scopus (113) Google Scholar). The vaccine quality depends to a considerable extent upon the chemical modifications caused by the formaldehyde treatment (1Rappuoli R. Levine M.M. Woodrow G.C. Kaper J.B. Cobon G.S. New Generation Vaccines. 2nd Ed. Marcel Dekker, Inc., New York1997: 417-435Google Scholar, 5Nencioni L. Volpini G. Peppoloni S. Bugnoli M. De Magistris T. Marsili I. Rappuoli R. Infect. Immun. 1991; 59: 625-630Crossref PubMed Google Scholar, 6Metz B. Jiskoot W. Hennink W.E. Crommelin D.J.A. Kersten G.F.A. Vaccine. 2003; 22: 156-167Crossref PubMed Scopus (72) Google Scholar). Formaldehyde is also used for isotope-labeling of proteins (7Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (172) Google Scholar, 8Means G.E. Feeney R.E. Anal. Biochem. 1995; 224: 1-16Crossref PubMed Scopus (65) Google Scholar, 9Gold T.B. Smith S.L. Digenis G.A. Pharm. Dev. Technol. 1996; 1: 21-26Crossref PubMed Scopus (25) Google Scholar), for studying protein-protein interactions, e.g. histone organization in nucleosomes (10Kunkel G.R. Mehrabian M. Martinson H.G. Mol. Cell. Biochem. 1981; 34: 3-13Crossref PubMed Scopus (63) Google Scholar, 11Jackson V. Cell. 1978; 15: 945-954Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 12Feldman M.Y. Prog. Nucleic Acid Res. Mol. Biol. 1973; 13: 131-149Google Scholar), and for fixation of cells and tissues (13Fox C.H. Johnson F.B. Whiting J. Roller P.P. J. Histochem. Cytochem. 1985; 33: 845-853Crossref PubMed Scopus (811) Google Scholar). Glutaraldehyde is utilized for the preparation of bioprostheses such as heart valves and vascular grafts (14Jayakrishnan A. Jameela S.R. Biomaterials. 1996; 17: 471-484Crossref PubMed Scopus (394) Google Scholar, 15Khor E. Biomaterials. 1997; 18: 95-105Crossref PubMed Scopus (346) Google Scholar, 16Mainil Varlet P. Rieser F. Grogan S. Mueller W. Saager C. Jakob R.P. Osteoarthritis Cartilage. 2001; 9: S6-S15Abstract Full Text PDF PubMed Scopus (115) Google Scholar) and for conjugation of enzymes to carrier systems (17Cabral J.M.S. Kennedy J.F. Taylor R.F. Protein Immobilization. Marcel Dekker, Inc., New York1991: 123-124Google Scholar). These examples demonstrate the wide range of roles of aldehydes in the biomedical field. Besides the use of aldehydes in diverse applications, they can also destroy important sites of proteins, such as crucial epitopes or active sites in enzymes. Several decades ago, extensive model studies were performed on reactions of formaldehyde with mixtures of amino acids and derivatives to determine which amino acids can cross-link (18Fraenkel-Conrat H. Olcott H.S. J. Biol. Chem. 1948; 174: 827-843Abstract Full Text PDF PubMed Google Scholar, 19Fraenkel-Conrat H. Olcott H.S. J. Amer. Chem. Soc. 1948; 70: 2673-2684Crossref PubMed Scopus (244) Google Scholar, 20Blass J. Bizzini B. Raynaud M. C. R. Acad. Sci. (Paris). 1965; 261: 1448-1449Google Scholar, 21Blass J. Bull. Soc. Chim. Fr. 1966; 10: 3120-3121Google Scholar). It was demonstrated that formaldehyde reacts first with the amino and thiol groups of amino acids and forms methylol derivatives. In the case of primary amino groups, the methylol groups partially undergo condensation to an imine, also called a Schiff-base (Scheme 1). Subsequently, the imine can cross-link with glutamine, asparagine, tryptophan, histidine, arginine, cysteine, and tyrosine residues. Some of the chemical structures of the proposed adducts have been elucidated by NMR (22Kelly D.P. Dewar M.K. Johns R.B. Wei-Let S. Yates J.F. Adv. Exp. Med. Biol. 1977; 86A: 641-647Crossref PubMed Scopus (21) Google Scholar). This knowledge, however, is not sufficient to predict all possible modifications in proteins that are induced by formaldehyde. Moreover, the formation of modifications is influenced by various factors, such as the rate of a particular cross-link reaction, the position and local environment of each reactive amino acid in the protein, the pH, the components present in the reaction solution, and the reactant concentrations. Importantly, the nature of all possible chemical modifications in proteins caused by formaldehyde has not yet been fully elucidated, in part because of the low resolution and sensitivity of the analytical methods available at the time the above studies were performed (18Fraenkel-Conrat H. Olcott H.S. J. Biol. Chem. 1948; 174: 827-843Abstract Full Text PDF PubMed Google Scholar, 19Fraenkel-Conrat H. Olcott H.S. J. Amer. Chem. Soc. 1948; 70: 2673-2684Crossref PubMed Scopus (244) Google Scholar, 20Blass J. Bizzini B. Raynaud M. C. R. Acad. Sci. (Paris). 1965; 261: 1448-1449Google Scholar, 21Blass J. Bull. Soc. Chim. Fr. 1966; 10: 3120-3121Google Scholar). However, the current availability of tandem high-performance liquid chromatography-mass spectrometry provides more detailed insight into the chemistry of protein-formaldehyde reactions. The purpose of this study was to elucidate the chemical nature of the reactions between formaldehyde and proteins. Therefore, a set of model peptides was prepared and used to map systematically the different chemical modifications induced by formaldehyde treatment. The selected peptides can be divided into two groups (see Table I): the first group had the amino acid sequence Ac-VELXVLL, in which one amino acid residue (X) varies and the remaining amino acid residues are non-reactive with formaldehyde. The second group was synthesized for studying the possible formation of intramolecular, formaldehyde-mediated cross-links between two reactive residues and contained peptides with the following sequence: Ac-LOENXLLZF-NH2, where O, X, and Z are either a (non-reactive) alanine residue or an arginine, lysine, and histidine residue in different permutations (see Table I). The reaction conditions were largely based on the detoxification process of diphtheria toxin for vaccine production (1Rappuoli R. Levine M.M. Woodrow G.C. Kaper J.B. Cobon G.S. New Generation Vaccines. 2nd Ed. Marcel Dekker, Inc., New York1997: 417-435Google Scholar). Because glycine is used as a reagent during the inactivation of diphtheria toxin by formaldehyde for the preparation of diphtheria toxoid vaccines (6Metz B. Jiskoot W. Hennink W.E. Crommelin D.J.A. Kersten G.F.A. Vaccine. 2003; 22: 156-167Crossref PubMed Scopus (72) Google Scholar), it was especially chosen to study in detail the cross-link reaction with peptides. The conversion of peptides was monitored by tandem reversed-phase liquid chromatography, electrospray ionization mass spectrometry (LC/MS). 1The abbreviations used are: LC/MS, tandem liquid chromatography-electrospray ionization mass spectrometry; Fmoc, N-(9-fluorenyl) methoxycarbonyl; Me2SO, dimethyl sulfoxide; TFA, trifluoroacetic acid; Ac-Arg-OME, Nα-acetylarginine methyl ester; MS, mass spectrometry. 1The abbreviations used are: LC/MS, tandem liquid chromatography-electrospray ionization mass spectrometry; Fmoc, N-(9-fluorenyl) methoxycarbonyl; Me2SO, dimethyl sulfoxide; TFA, trifluoroacetic acid; Ac-Arg-OME, Nα-acetylarginine methyl ester; MS, mass spectrometry. In this paper, we present an overview of the major conversion products resulting from reactions between model peptides and formaldehyde (in the absence and presence of glycine), several of which have not been can be used for the and of reactive sites in proteins after to in this study and mass after formaldehyde treatment was not peptides and could not be in Therefore, the peptides and were peptides and could not be in Therefore, the peptides and were products with mass were formed after incubation with formaldehyde by incubation with products with mass were formed after incubation with formaldehyde by incubation with products with mass were formed after incubation with formaldehyde by incubation with products with mass were formed after incubation with formaldehyde by incubation with was not The peptides and could not be in Therefore, the peptides and were products with mass were formed after incubation with formaldehyde by incubation with in a new acid and were from The cyanoborohydride was from The methyl was from was from The was from The were synthesized on a by an peptide with a reaction were performed with acid and were performed in and from The group was with and from the was with trifluoroacetic acid for and which were treated with The peptides were by reversed-phase liquid and was by peptides were in or to a of with the reaction of peptides with formaldehyde, of a peptide solution, of and of a second agent Ac-Arg-OME, or were added to of The reaction was by of an of formaldehyde. the was for at were at in of different reaction conditions was by the reaction pH, reagent and the of of The reaction of peptides with formaldehyde and glycine was monitored for were after and and and and at To the of pH, reactions were performed in at and The of the of the reagents on formation was by the formaldehyde or the glycine to of and To determine cross-links in was added after formaldehyde of of formaldehyde was performed on a high-performance liquid with a with The was with to a peptide of and of the was on the The was for with in at a rate of to formaldehyde. The peptide was by a from in in The containing the peptide was in a and in of was at Formaldehyde of and an arginine Ac-Arg-OME, were or in to of reaction was prepared by of glycine solution, of solution, and of formaldehyde to of each the was by The preparation was for at was at by were by of peptide solution, of of solution, and of by incubation for at Subsequently, were at were to a of in containing and with an of was with of the was by an of the and the electrospray to The was to were from by of to The were for each mass were analyzed by to electrospray mass spectrometry as by E. A. J. Sci. 2002; Scopus Google Scholar). each peptide was to a of in containing and of was used for To the for were on a with at a rate of and by acid in as for were by reversed-phase by a analytical with at a rate between was from acid in to in the were in for The were in the to determine the mass and conversion of peptides after incubation with formaldehyde. The was set to and electrospray was set to second was performed to detailed sequence Therefore, the peptides were analyzed by an by of the in the The was set on of set of peptides was used to the reactivity of amino acid residues with formaldehyde. The reaction was monitored a by In of the peptides to formaldehyde in a reaction time of all modifications that were in this study were by in the that reactions did not and that a conversion rate was above the conversion of the peptides was with the reactant on we used the following reaction conditions in the of this study excess of formaldehyde with to the peptide incubation at and for of and was with amino acids residues, which were not to react with formaldehyde I). that peptide was not after incubation with formaldehyde. the peptides containing a arginine and tryptophan histidine or lysine residue products with a mass of second in peptides containing a tryptophan or a lysine residue and was This caused a mass of and in with the of peptide and peptide mass of formaldehyde treatment of peptide and peptide did not of reaction a could be attached to the in peptide and the in peptide could react with formaldehyde and residues were reactive with formaldehyde (see Table I). Therefore, we that the reaction the methylol and imine depends upon the amino acid The of is an of the formation of a methylol group (Scheme reaction 1). reaction the conversion between upon the The formation of a methylol to peptides is a reaction, because the conversion of the peptide was to after of free formaldehyde. of were performed on peptides to that the methylol was on cysteine, histidine, lysine, and arginine residues and The that peptide were present with a mass of they contained a cysteine, arginine, or a histidine peptide residues had the mass as the of peptides. on the peptide containing a methylol on the lysine residue peptide with a mass of of because of of the methylol group (Scheme reaction The methylol on a tryptophan residue could not be by because of the low conversion In conclusion, of cysteine, histidine, lysine, arginine, and tryptophan residues can methylol groups in the presence of formaldehyde. Two possible reaction products could for a mass of found in peptides and the formation of an imine or a methylene (Scheme reactions and These were by by as a of peptide Inc., Scholar). performed on peptide that the mass of was on the tryptophan residue. The of tryptophan was after the reaction with formaldehyde and a new that the tryptophan residue was new were the that cross-links were formed between two residues. The proposed of the tryptophan residue is in were also performed on peptide to determine the of formed. The during formaldehyde a with a mass of and a of The of can be to an of an lysine residue. of was not in this is the of which the The of which was found after formaldehyde is of the formation of a Schiff-base second of the presence of a Schiff-base in peptide was the reaction with which was added after the incubation with formaldehyde. The group of lysine was to a with a mass of (Scheme In conclusion, of tryptophan and lysine residues can during incubation with of primary amino groups by formaldehyde and (7Jentoft N. Dearborn D.G. Methods Enzymol. 1983; 91: 570-579Crossref PubMed Scopus (172) Google Scholar, 8Means G.E. Feeney R.E. Anal. Biochem. 1995; 224: 1-16Crossref PubMed Scopus (65) Google containing a free amino group was into an with a mass of to the formaldehyde can a methylene in such as by NMR and R.B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. J. Biochem. 1996; PubMed Scopus Google Scholar, E. M. 2001; PubMed Scopus Google Scholar). The resulting is a (Scheme reaction 1). To the formation of a we added to the peptide after incubation with formaldehyde. This in the formation of a peptide with a mass of (Scheme reaction that an had formed. formaldehyde and N-terminal amino groups are to a a mass of was then shown for peptide The formation of cross-links was also for peptides because they two or amino acid residues that are reactive with they lysine, arginine, histidine residues. reaction peptide one with a mass of after formaldehyde treatment. This that one methylol was on the arginine or the histidine residue. with a mass of was also could not be the formaldehyde to a reaction was with a mass of that two methylol groups were attached to the However, cross-link was formed in this because in that a mass of was Besides the with a mass of peptide two products each with a mass of These products be to the formation of a Schiff-base on the lysine residue or a methylene between lysine and histidine residues. of to the peptide two of a peptide with a mass of and a with an of The mass of can be by the formation of lysine, the of a with an cross-link between the lysine and the histidine residue. peptides and two of adducts with a mass of These products could not be by which that two methylene bridges had been formed between the of lysine and The proposed structures are in The following was performed to this a acid residue between the arginine and the lysine residue which of the peptides by the and the peptide were with The peptide was by two with of and the the peptide with a mass of was partially to a with a mass of The mass of that the peptide was at the of the acid residue and that the two were to each by means of cross-links between the lysine and arginine residues. This the proposed structures in In to studies with amino acids (18Fraenkel-Conrat H. Olcott H.S. J. Biol. Chem. 1948; 174: 827-843Abstract Full Text PDF PubMed Google Scholar, 19Fraenkel-Conrat H. Olcott H.S. J. Amer. Chem. Soc. 1948; 70: 2673-2684Crossref PubMed Scopus (244) Google Scholar), peptides were not This be because of in reaction conditions and possible reactivity of peptides as with free amino the of the peptides did not us to cross-linking between two peptides at concentrations. reaction time or peptides with different reactive residues did not in intermolecular between and the it was shown that cross-links were formed between lysine and histidine residues or between lysine and arginine residues after formaldehyde treatment. To formaldehyde can methylene bridges between amino acid residues, all peptides were for with a excess of formaldehyde and that glycine and formaldehyde reacted to a reactive imine with an mass of This reacted with peptides containing a histidine and or tryptophan residue products with a mass of This can be by the coupling of glycine to the peptides via a methylene structures of adducts attached to different amino acid residues present in peptides in a new The peptide with a tyrosine residue two products with an of and which means that one or two adducts were to the to the of the group This of reaction is known as the J. Bull. Soc. Chim. Fr. 1966; 10: 3120-3121Google Scholar, J.F. Formaldehyde, Ed. New Scholar). was also by formaldehyde to the N-terminal amino group of peptide which the peptide a mass of The formation of this in two steps: (i) a is formed and then (ii) the glycine is attached via a methylene to this The proposed is in reaction peptides were also by formaldehyde and products were found with mass of and The mass can be by the coupling of one or two glycine molecules to the peptide via two methylene bridges (see Table To that this on the arginine an arginine Ac-Arg-OME, was treated with formaldehyde and The two products from had the mass of and proposed structures are in were performed on products with of and The with mass of was after four into with of and The with mass of was to with of and The possible structures of the are in formaldehyde did not cross-link of glycine to peptides and containing a or a lysine of cross-links have been in several (1Rappuoli R. Levine M.M. Woodrow G.C. Kaper J.B. Cobon G.S. New Generation Vaccines. 2nd Ed. Marcel Dekker, Inc., New York1997: 417-435Google Scholar, 8Means G.E. Feeney R.E. Anal. Biochem. 1995; 224: 1-16Crossref PubMed Scopus (65) Google Scholar, D.P. Dewar M.K. Johns R.B. Wei-Let S. Yates J.F. Adv. Exp. Med. Biol. 1977; 86A: 641-647Crossref PubMed Scopus (21) Google Scholar, W. 70: PubMed Scopus Google Scholar, J. Chem. Soc. PubMed Scopus Google Scholar). the reaction time or the formaldehyde and glycine did not have The peptide were analyzed by in an Because the methylene between two amino groups be in an environment J. Bizzini B. Raynaud M. (Paris). Google Scholar), the were also at by ionization modifications were found in peptides. These are in to the of treated and with formaldehyde. bridges were formed between the two amino groups in and between the amino and thiol groups of (22Kelly D.P. Dewar M.K. Johns R.B. Wei-Let S. Yates J.F. Adv. Exp. Med. Biol. 1977; 86A: 641-647Crossref PubMed Scopus (21) Google Scholar, J. Chem. Soc. PubMed Scopus Google Scholar). However, and formed in intermolecular cross-links have to be formed between the peptide and studies with a peptide containing two lysine residues upon different of peptides treated with formaldehyde and glycine demonstrated that several products were formed with different I). These peptides two or residues that can react with formaldehyde. The can be as a of methylol and and intermolecular The conversion of peptides after treatment predict the reaction rate of cross-links in proteins are by the reaction of formaldehyde with lysine residues. Subsequently, the adducts cross-links with reactive residues in environment and especially with the residues that have the Therefore, the conversion of peptides was after incubation with formaldehyde and glycine The that the was rapidly attached to free N-terminal amino groups, to arginine and tyrosine residues of and to a lesser extent to asparagine, glutamine, histidine, and tryptophan of peptides to a after incubation with amino in a new between and the cross-link reactions between glycine and peptides caused by formaldehyde can via an To peptides and containing a histidine, lysine, arginine, and tryptophan were with formaldehyde and for was to peptide and the peptide a mass of This that two methylene bridges were formed between the primary group of the lysine residue in peptide and the arginine containing a histidine, arginine, or a tryptophan residue did not react with formaldehyde and Ac-Arg-OME, that the reaction occurred by means of a peptide with a residue was treated with formaldehyde and to demonstrate that thiol groups can also cross-links with arginine residues. with mass of was that one methylene had been formed between the thiol group of peptide and the arginine It has been in the that formaldehyde can react with the group and group of N-terminal formation of derivatives E. A. J. Chem. Soc. Chem. Scopus Google Scholar, J. Protein Res. PubMed Scopus Google Scholar). and present we that residues can cross-links at with and with N-terminal amino groups as a of formaldehyde treatment. This study has demonstrated on sequence, peptides undergo a of chemical modifications after formaldehyde treatment. The modifications can be divided into (i) methylol groups, (ii) Schiff-bases, and methylene bridges. The formation of methylol and is and are to methylol and Schiff-base derivatives could be demonstrated in several peptides by were on residues with an amino or a thiol