Mechanistic insights into the multistage gas-phase fragmentation behavior of phosphoserine- and phosphothreonine-containing peptides

The increasing use of multistage tandem mass spectrometry (MS/MS and MS (3)) methods for comprehensive phosphoproteome analysis studies, as well as the emerging application of in silico spectral intensity prediction algorithms in enhanced database search analysis strategies, necessitate the development of an improved understanding of the mechanisms and other factors that affect the gas-phase fragmentation reactions of phosphorylated peptide ions. To address this need, we have examined the multistage collision-induced dissociation (CID) behavior of a set of singly and doubly charged phosphoserine- and phosphothreonine-containing peptide ions, as well as their regioselectively or uniformly deuterated derivatives, in a quadrupole ion trap mass spectrometer. Consistent with previous reports, the neutral loss of phosphoric acid (H 3PO 4) was observed as a dominant reaction pathway upon MS/MS. The magnitude of this loss was found to be highly dependent on the proton mobility of the precursor ion for both phosphoserine- and phosphothreonine-containing peptides. In contrast to that currently accepted in the literature, however, the results obtained in this study unequivocally demonstrate that the loss of H 3PO 4 does not predominantly occur via a "charge-remote" beta-elimination reaction. The observation of product ions corresponding to the loss of formaldehyde (CH 2O, 30 Da, or CD 2O, 32 Da) or acetaldehyde (CH 3CHO, 44 Da) upon MS (3) dissociation of the [M+ nH-H 3PO 4] ( n+ ) product ions from phosphoserine- and phosphothreonine-containing peptide ions, respectively, provide experimental evidence for a "charge-directed" mechanism involving an S N2 neighboring group participation reaction, resulting in the formation of a cyclic product ion. Potentially, these "diagnostic" MS (3) product ions may provide additional information to facilitate the characterization of phosphopeptides containing multiple potential phosphorylation sites.     

The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite

Both peroxynitrous acid and peroxynitrite react with methionine, k(acid) = (1.7 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 8.6 +/- 0.2 M(-1) s(-1), respectively, and with N-acetylmethionine k(acid) = (2.8 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 10.0 +/- 0.1 M(-1) s(-1), respectively, to form sulfoxides. In contrast to the results of Pryor et al. (1994, Proc. Natl. Acad. Sci. USA 91, 11173-11177), a linear correlation between k(obs) and [met] was obtained. Surprisingly, for every two sulfoxides and nitrites formed, one peroxynitrite is converted to nitrate. Thus, methionine also catalyzes the isomerization of peroxynitrite to nitrate. Neither the pH nor the concentration of methionine affected the distribution of the yields of nitrite, nitrate, and methionine sulfoxide, which were the only products detected. No products other than nitrite, nitrate, and methioninesulfoxide could be detected. The reactions of methionine and N-acetylmethionine with peroxynitrous acid and peroxynitrite are simple bimolecular reactions that do not involve an activated form of peroxynitrous acid or of peroxynitrite. Nitrite, produced together with methionine sulfoxide, or present as a contamination in the peroxynitrite preparation, is not innocuous, but oxidizes methionine by one electron, which leads to the formation of methional and ethylene.     

Reduction of methionine sulfoxide to methionine by Escherichia coli.

L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.     

Synthetic approaches to peptides containing the L-Gln-L-Val-D(S)-Dmt motif

The pseudoprolines S-Dmo (5,5-dimethyl-4-oxaproline) and R-Dmt (5,5-dimethyl-4-thiaproline) have been used to study the effects of forcing a fully cis conformation in peptides. Synthesis of peptides containing these (which have the same configuration as L-Pro) is straightforward. However, synthesis of peptides containing S-Dmt is difficult, owing to the rapid cyclisation of L-Aaa-S-Dmt amides and esters to form the corresponding diketopiperazines (DKP); thus the intermediacy of L-Aaa-S-Dmt amides and esters must be avoided in the synthetic sequence. Peptides containing the L-Gln-L-Val-D(S)-Dmt motif are particularly difficult, owing to the insolubility of coupling partners containing Gln. Introduction of Gln as N-Boc-pyroglutamate overcame the latter difficulty and the dipeptide active ester BocPygValOC(6)F(5) coupled in good yield with S-DmtOH. BocPygVal-S- DmtNH(CH(2))(2)C(6)H(4)NO(2) was converted quantitatively to BocGlnVal-S-DmtNH(CH(2))(2)C(6)H(4)NO(2) with ammonia, demonstrating the utility of this approach. Two peptide derivatives (CbzSerLysLeuGlnVal-S-DmtNH(CH(2))(2)C(6)H(4)NO(2) and CbzSerSerLysLeuGlnVal-S- DmtNH(CH(2))(2)C(6)H(4)NO(2)) were assembled, using these new methods of coupling a dipeptide acid active ester with S-DmtOH and introduction of Gln as Pyg, followed by conventional peptide couplings. The presence of the Val caused these peptides to be cleaved very slowly by prostate-specific antigen (PSA) at Leu Gln, rather than the expected Gln Val.     

Current mechanistic approaches to the chemoprevention of cancer

The prevention of cancer is one of the most important public health and medical practices of the 21st century. We have made much progress in this new emerging field, but so much remains to be accomplished before widespread use and practice become common place. Cancer chemoprevention encompasses the concepts of inhibition, reversal, and retardation of the cancer process. This process, called carcinogenesis, requires 20-40 years to reach the endpoint called invasive cancer. It typically follows multiple, diverse and complex pathways in a stochastic process of clonal evolution. These pathways appear amenable to inhibition, reversal or retardation at various points. We must therefore identify key pathways in the evolution of the cancer cell that can be exploited to prevent this carcinogenesis process. Basic research is identifying many genetic lesions and epigenetic processes associated with the progression of precancer to invasive disease. Many of these early precancerous lesions favor cell division over quiescence and protect cells against apoptosis when signals are present. Many oncogenes are active during early development and are reactivated in adulthood by aberrant gene promoting errors. Normal regulatory genes are mutated, making them insensitive to normal regulatory signals. Tumor suppressor genes are deleted or mutated rendering them inactive. Thus there is a wide range of defects in cellular machinery which can lead to evolution of the cancer phenotype. Mistakes may not have to appear in a certain order for cells to progress along the cancer pathway. To conquer this diverse disease, we must attack multiple key pathways at once for a predetermined period of time. Thus, agent combination prevention strategies are essential to decrease cancer morbidity. Furthermore, each cancer type may require custom combination of prevention strategies to be successful.     

Analyses of methionine sulfoxide reductase activities towards free and peptidyl methionine sulfoxides

There have been insufficient kinetic data that enable a direct comparison between free and peptide methionine sulfoxide reductase activities of either MsrB or MsrA. In this study, we determined the kinetic parameters of mammalian and yeast MsrBs and MsrAs for the reduction of both free methionine sulfoxide (Met-O) and peptidyl Met-O under the same assay conditions. Catalytic efficiency of mammalian and yeast MsrBs towards free Met-O was >2000-fold lower than that of yeast fRMsr, which is specific for free Met-R-O. The ratio of free to peptide Msr activity in MsrBs was 1:20-40. In contrast, mammalian and yeast MsrAs reduced free Met-O much more efficiently than MsrBs. Their k(cat) values were 40-500-fold greater than those of the corresponding MsrBs. The ratio of free to peptide Msr activity was 1:0.8 in yeast MsrA, indicating that this enzyme can reduce free Met-O as efficiently as peptidyl Met-O. In addition, we analyzed the in vivo free Msr activities of MsrBs and MsrAs in yeast cells using a growth complementation assay. Mammalian and yeast MsrBs, as well as the corresponding MsrAs, had apparent in vivo free Msr activities. The in vivo free Msr activities of MsrBs and MsrAs agreed with their in vitro activities.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Functions and evolution of selenoprotein methionine sulfoxide reductases

Methionine sulfoxide reductases (Msrs) are thiol-dependent enzymes which catalyze conversion of methionine sulfoxide to methionine. Three Msr families, MsrA, MsrB, and fRMsr, are known. MsrA and MsrB are responsible for the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide residues in proteins, respectively, whereas fRMsr reduces free methionine-R-sulfoxide. Besides acting on proteins, MsrA can additionally reduce free methionine-S-sulfoxide. Some MsrAs and MsrBs evolved to utilize catalytic selenocysteine. This includes MsrB1, which is a major MsrB in cytosol and nucleus in mammalian cells. Specialized machinery is used for insertion of selenocysteine into MsrB1 and other selenoproteins at in-frame UGA codons. Selenocysteine offers catalytic advantage to the protein repair function of Msrs, but also makes these proteins dependent on the supply of selenium and requires adjustments in their strategies for regeneration of active enzymes. Msrs have roles in protecting cellular proteins from oxidative stress and through this function they may regulate lifespan in several model organisms.     

The enzymology and biochemistry of methionine sulfoxide reductases

The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.     

Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages

Molecular Biology Reports - Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the...     

Wheat methionine sulfoxide reductase genes and their response to abiotic stress

The wheat cultivar Shanrong no. 3 (cv. SR3) tolerates both salinity and drought stress more effectively than does its progenitor cultivar Jinan 177 (cv. JN177). When the cultivars are subjected to stress, a number of genes encoding methionine sulfoxide reductase (MSRs) are known to be upregulated in SR3. Here, a set of 12 full length Triticum aestivum MSR (TaMSR) cDNAs have been isolated from cv. SR3. The genes were transcribed in the wheat root, stem, and leaf in plants sampled at various developmental stages. Those induced by salinity and drought harbored known stress-responsive cis elements in their promoter region. The constitutive expression in Arabidopsis thaliana of four MSRs which were induced by salt and drought in microarray assay showed that the product of one (TaMSRA2) heightened the plant’s tolerance to NaCl, methylviologen (MV), and abscisic acid, that of the second (TaMSRA5) enhanced salinity tolerance, that of the third (TaMSRB1.1) increased tolerance to salinity, MV and H₂O₂, and that of the fourth (TaMSRB5.1) increased tolerance to both salinity and mannitol. The effect of the presence in A. thaliana of TaMSRB1.1 was to suppress the accumulation of reactive oxygen species and to increase the intracellular content of soluble sugars.     

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro

Summary.  An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO₂). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39°C for 12 h with or without MSO (1 mM) or MSO₂ (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 μmol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO₂ and 2-aminobutyric acid (2AB) in B and BP. MSO₂ as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO₂; but MSO can be utilized by B and BP for producing Met. Received December 28, 2001 Accepted May 21, 2002 Published online October 14, 2002 Acknowledgements The authors are extremely grateful to Professor H. Ogawa, the University of Tokyo, Japan and Dr. Takashi Hasegawa, Miyazaki University, Japan for inserting permanent rumen fistulae in goats. We would like to thank MONBUSHO for the award of a research scholarship to Mamun M. Or-Rashid since 1996–2001. Authors' address: Shaila Wadud, Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science, Miyazaki University, Miyazaki 889-2192, Japan, Fax. +81-985-58-7201, E-mail: rafatkun@hotmail.com     

Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [Me-13C, Me-2H(3)]methionine sulfoxide and [Me-13C, Me-2H(3)]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0 +/- 1.0 microM (means +/- SD, n = 8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.