Martin D. Shetlar

Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry

Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO2−). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing 3-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laser-induced photochemical decomposition. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed site-specific identification of 3-nitrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides.

Photochemical addition of amino acids to thymine.

Summary The ultraviolet light induced photoreactivity of thymine with the twenty two common amino acids has been surveyed. Lysine, arginine, cysteine, and cystine have been found to be reactive in forming heteroadducts. The results suggest that lysine and arginine may be involved in the experimentally observed ultraviolet induced crosslinking of histones to DNA in DNA-histone complexes.

PHOTOCHEMICAL ADDITION OF AMINO ACIDS AND PEPTIDES TO DNA

Abstract— The quantum yields for photochemical addition of twenty of the amino acids commonly occurring in proteins to denatured calf thymus DNA have been determined in deoxygenated phosphate buffer at λ 254 nM and pH 7 using a fluorescamine assay technique. Fifteen were found to be reactive, with cysteine, lysine, phenylalanine, tryptophan and tyrosine being the most reactive. Alanine, aspartic acid, glutamic acid, serine and threonine were unreactive. Analogous quantum yields for a series of eighteen peptides of the form glycyl X (X being one of the commonly occurring amino acids) were also determined, along with the corresponding quantum yields for L‐alanyl‐L‐alanine, L‐alanyl‐L‐tryptophan, L‐seryl‐L‐seryl‐L‐serine, L‐threonyl‐L‐threonyl‐L‐threonine, and L‐cystine‐bis‐glycine. All of the peptides were found to be reactive. The modified amino acids Nε‐methyllysine, Nε, Nε, Nε‐trimethyllysine and Nε‐acetyllysine, all occurring in minor amounts in the histone group of chromosomal proteins, were also found to be reactive as was Nα‐acetyllysine. The quantum yields for photoaddition of a selected group of amino acids and peptides to denatured DNA and native DNA are compared. In some cases higher quantum yields for photoaddition to denatured DNA are observed while in other cases the reverse is true. The effect of oxygen on the quantum yields for photoaddition of selected peptides to DNA was examined. While for most systems studied the amount of reaction in aerated systems was less than in deoxygenated systems, in the case of glycyl‐L‐phenylalanine the reverse was true.

The photochemistry of p-aminobenzoic acid.

Abstract— We have studied the photoreactions occurring when p‐aminobenzoic acid (PABA), a component of some sunscreens, is irradiated in aqueous solution. These studies were carried out in the presence and absence of oxygen, using light of Λ= 254 nm as well as light of wavelengths greater than 290 nm. In deoxygenated solution between pH 7.5 and 11.0, we found two photoproducts that were identified as 4‐(4′‐aminophenyl)aminobenzoic acid (I) and 4‐(2′‐amino‐5′‐carboxy‐phenyl)aminobenzoic acid (V); we used H and 13C NMR, electron impact mass spectrometry and synthesis by an independent route to identify each of these compounds. Rapid discoloration of the photolyzed sample was observed when PABA was irradiated in aerated solution. Although a number of products were detected under these conditions, the three most abundant stable compounds have been isolated and identified as 4‐amino‐3‐hydroxybenzoic acid, 4‐aminophenol and 4‐(4′‐hydroxyphenyl)aminobenzoic acid (IV). The latter compound was shown to result from rapid photo‐induced oxidation of I in the presence of oxygen. Even in the presence of trace amounts of oxygen, the yield of I was significantly reduced in favor of IV. Studies of the thermal oxidation of I, coupled with evidence gathered from studies of the photochemistry of incompletely deoxygenated PABA solutions, indicate that 4‐(2,5‐cyclohexadien‐4‐one)iminobenzoic acid (III) is an intermediate on the pathway between I and IV.

Photochemical addition of amino acids and peptides to polyuridylic acid.

Abstract— The quantum yields for photochemical addition of glycine and the L‐amino acids commonly occurring in proteins (excluding proline) to polyuridylic acid have been determined in deoxygenated phosphate buffer at pH 7, using a fluorescamine assay technique. All twenty amino acids were found to be reactive, with cysteine, tryptophan, phenylalanine, tyrosine, arginine, lysine and methionine being the most reactive. The analogous quantum yields for a series of eighteen dipeptides of the form glycyl X (X being one of the commonly occurring amino acids, including proline), of L‐alanyl‐L‐tryptophan, of the tripeptides L‐seryl‐L‐seryl‐L‐serine and L‐threonyl‐L‐threonyl‐L‐threonine, of the tetrapeptide L‐cystine‐bis‐glycine, and of the lysine derivative Nα‐acetyllysine were also measured. All were found to be reactive toward photoaddition to poly U.

Photochemical addition of amino acids and peptides to homopolyribonucleotides of the major DNA bases.

Abstract— The photochemical quantum yields for addition of glycine and the L‐amino acids commonly occurring in proteins (excluding proline) to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer at Λ 254 nm and pH 7, using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid.

PHOTOADDITION OF p-AMINOBENZOIC ACID TO THYMINE AND THYMIDINE

Abstract— Several studies in the literature have shown that DNA is damaged after UV irradiation in the presence of the sunscreen agent p‐aminobenzoic acid (PABA), both in vivo and in vitro. One type of damage has been shown to be the result of increased yields of pyrimidine cyclobutane dimer formation. However, it has been suggested that other types of lesions are produced as well. We have studied the photochemistry of the thymine‐PABA and thymidine‐PABA systems and report here the isolation and characterization of thymine‐PABA and thymidine‐PABA photoadducts. These products have been identified, respectively, as 5‐(2‐amino‐5‐carboxyphenyl)‐5,6‐dihydrothymine and isomeric forms of 5‐(2‐amino‐5‐carboxyphenyl)‐5,6‐dihydrothymidine. The quantum yields for the formation of these adducts in deaerated aqueous solutions at pH 7.0 have been determined to be 9.5 × 10–4 and 4.3 × 10–3 for the thymine and thymidine based adducts respectively. A pH profile for the thymine‐PABA system indicated a maximum quantum yield for adduct formation at pH 6.5, although it could be detected over the whole pH range studied (pH 3.5–11.0).

The Photochemistry of Thymidylyl‐(3′‐5′)‐5‐methyl‐2′‐deoxycytidine in Aqueous Solution¶

The photochemistry of the dinucleoside monophosphate thymidylyl‐(3′‐5′)‐5‐methyl‐2′‐deoxycytidine (Tpm5dC) has been studied in aqueous solution using both 254 nm and UV‐B radiation. A variety of dinucleotide photoproducts containing 5‐methyl‐cytosine (m5C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis‐syn [c,s] and transsyn forms), a (6–4) adduct and its realted Dewar isomer, and two isomers of a product in which the m5C moiety was converted into an acrylamidine. Small amounts of thymidylyl‐(3′‐5′)‐thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m5C moiety was lost, thus generating 3′‐thymidylic acid esterified with 2′‐deoxyribose at the 5‐hydroxyl on the sugar mioety. The c,s CBD of Tpm5dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and −103.5J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm5dC.

THE PHOTOREACTIONS AND FREE RADICAL INDUCED REACTIONS OF 1,3‐DIMETHYLURACIL IN METHANOL AND OTHER ALCOHOLS

Abstract— Photoexcited 1,3‐dimethyluracil (DMU) reacts with methanol to give 1,3‐dimethyl‐6‐hydroxymethyl‐5‐hydrouracil (1a) in addition to cyclobutane type dimers and 1,3‐dimethyl‐6‐methoxy‐5‐hydrouracil (2a). Free radical induced reaction, initiated by photodecomposition of di‐t‐butyl peroxide with light of wavelength greater than 290nm, leads to 1a, 5,6‐dihydroxymethyl‐5,6‐dihydro‐1,3‐dimethyl‐uracil (1d) and 6‐hydroxymethyl‐1,3‐dimethyluracil as the products.

Formation of thymine-lysine adducts in irradiated DNA-lysine systems

Summary Thymine, contained in DNA excited by ultraviolet light (λ > 260 nm), adds lysine to form an acid stable thymine-lysine adduct.