Quantitation of methylated hemoglobin adducts in a signature peptide from rat blood by liquid chromatography/negative electrospray ionization tandem mass spectrometry

Hemoglobin adducts are often used as biomarkers for exposure to reactive chemicals in toxicology studies. Therefore, fast, sensitive, accurate, and reproducible methods for quantifying these protein adducts are key to evaluate test material dosimetry. A methodology has been developed for the quantitation of methylated hemoglobin adducts isolated from rats exposed to the model alkylating agent: methyl methane sulfonate (MMS). After 4 days of MMS exposure by oral gavage, hemoglobin was isolated from rat blood and digested with trypsin. The tryptic digestion solution was used for the adducted hemoglobin signature peptide quantitation via liquid chromatography/negative tandem mass spectrometry (LC/ESI-MS/MS). The limit of quantitation (LOQ) for the methylated hemoglobin beta chain N-terminal signature peptide (MeVHLTDAEK) was 1.95 ng/mL (5.9 pmol/mg globin). The calibration curves were linear over a concentration range of 1.95 to 625 ng/mL, with a correlation coefficient R2 >0.998, accuracy of 85.8 to 119.3%, and precision of 0.9 to 19.4%.     

Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive method has been developed for the direct quantitation of the methyl phosphotriester DNA adduct of thymidyl(3'-5')thymidine (dTp(Me)dT) from enzymatic DNA hydrolysates prepared from cultured cells treated with low levels of N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS), by rapid and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). The lower limit of quantitation was 0.1 ng/mL (6.4 adducts per 10(8) nucleotides). Linearity of the calibration curve was greater than 0.999 from 0.1 to 50 ng/mL. Intra-day precision for three levels of quality control samples ranged from 4.27 to 15.62%. Interday precision ranged from 2.46 to 11.95%. Using this method, the levels of dTp(Me)dT in DNA enzymatic hydrolysates obtained from a series of incubations of mouse lymphoma cells with low doses of MNU (50 microM) were quantified.     

Quantitation of DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative electrospray tandem mass spectrometry

A rapid and selective method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the direct quantitation of a methyl phosphotriester DNA adduct, thymidyl (3'-5') thymidine [dTp(Me)dT] from enzymatic hydrolysates of DNA (either in vitro DNA or in cell culture) treated with MNU (N-methyl-N-nitrosourea) or MMS (methyl methane sulfonate). The lower limit of quantitation was 2 ng/mL. Linearity of the calibration curve was greater than 0.999 from 2 to 1000 ng/mL. Intraday precision for four levels of quality controls ranged from 2.8 to 20.1%, and interday precision ranged from 2.9 to 5.6%. This method was used to quantify the levels of dTp(Me)dT in enzymatic hydrolysates of DNA obtained from a series of incubations of salmon testis DNA or mouse lymphoma cells with either MNU or MMS.     

Desymmetrisation of a diallyl system by intramolecular Heck reaction

The development of a desymmetrising cyclisation of a benzylic diallyl iodide is reported. The final procedure generates a new quaternary chiral centre in greater than 80% yield giving a highly functionalised methylene indan product suitable for further application in synthesis.     

Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro

The reactions of two model mutagenic and carcinogenic alkylating agents, N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a 'super clastogen' while MNU is a 'super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects.     

Mechanism of cerium (IV) oxidation of glucose and cellulose

Cerium (IV) oxidations of model compounds for the hydroxylic functional groups of cellulose were conducted in 1.0M perchloric acid. Glucose, the model selected for the reducing end group, was oxidized 360 times faster than Schardinger β-dextrin, the model for anhydro-D -glucose repeating units. In the presence of a fourfold excess of glucose, stoichiometry indicated specific conversion to arabinose; the competitive oxidation of arabinose produced is insignificant. Specific C₁–C₂ bond cleavage was also indicated for 2-O-methyl-D -glucose, galactose, 2-O-methyl-D -galactose, and cellobiose. Anhydro-D -glucose units were oxidized predominantly by C₂–C₃ bond cleavage as shown by the identification of erythrose and glyoxal in hydrolyzates of cerium (IV) oxidized Schardinger β-dextrin and cellulose. Kinetic studies showed that chelate complexes were involved in oxidations of glucose, methyl β-D -glucopyranoside, 1,5-anhydro-D -glucitol, and Sahardinger β-dextrin. The oxidations of glucose derivatives which differed with respect to substituents on C₁ and C₂ demonstrated the importance of the hemiacetal group and the presence of oxygen on C₂. For example, the relative rates of oxidation at 15°C for methyl β-D -glucopyranoside, 1,5-anhydro-D -glucitol, 2-deoxy-D -glucose, glucose, and 2-O-methyl-D -glucose are 1:1:12:360:1860, respectively. The mechanism of glucose oxidation is thought to involve formation of a chelate complex, disproportionation of the complex to form a free radical at either C₁ or C₂ and further rapid oxidation to 4-O-formyl-D -arabinose which is hydrolyzed in the reaction medium. General implications of the experimental results pertaining to initiation of vinyl graft polymerization on cellulose are discussed.     

Schwefel in metallurgisehen Produkten

Analytical and Bioanalytical Chemistry -