Selective production of rat mutant selenoprotein W with and without bound glutathione.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.

An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or nano-ESI mass spectrometry for proteolytic digests.

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2-3 microL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.

Purification, characterization, and glutathione binding to selenoprotein W from monkey muscle.

Selenoprotein W was purified from monkey skeletal muscle to investigate its binding of glutathione. The purification was accomplished by concentration of the cytosol with an Amicon cell, gel filtration using Sephadex G-50, cation-exchange chromatography with CM-Sephadex, and reverse-phase high-pressure liquid chromatography using a C-18 Vydac column. Selenoprotein W was monitored during purification by slot blots. These steps resulted in an electrophoretically pure selenoprotein W preparation that was estimated by gel filtration to have molecular weight of about 10 kDa. N-terminal amino acid sequencing was used to confirm that the pure proteins were selenoprotein W. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) revealed that the proteins existed in three masses of 9635 +/- 7, 9371 +/- 11, and 9330 +/- 5 Da. The theoretical mass of the protein predicted from the cDNA sequence is 9330 Da. The 9635-Da form of the protein was shown to contain bound glutathione (306 Da), which could be released by reduction with dithiothreitol at 50 degreesC. The form with a mass of 9371 Da is assumed to result from binding of an unidentified 41-Da moiety to the 9330-Da form of the protein. MALDI peptide mapping with endoproteinase Glu-C suggested that glutathione is bound to the 36th amino acid (cysteine) of selenoprotein W.

Selenoprotein W of rat muscle binds glutathione and an unknown small molecular weight moiety.

When purified from rat muscle, selenoprotein W is fractionated into four forms distinguished by slightly different chromatographic behavior. Precise masses of the four forms were determined by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. The mass distribution of the forms (9549, 9592, 9853, and 9898 d) suggests that they occur through derivatization of the lowest mass form with two moieties of approximate masses 44 and 305 d. The apparent 305 d moiety was demonstrated to be glutathione (307 d) by reductive release from the 9853 d protein form with 1000-fold excess of dithiothreitol at 50 degrees C. Milder conditions failed to remove the glutathione. The reduction produced nearly stoichiometric amounts of free glutathione as determined by HPLC of a fluorometric derivative. HPLC retention of the protein changed to match that of the 9549 d form, and a change of mass to 9550 d was observed by MALDI mass spectrometry. The identity of the 44 d moiety is unknown. The presence of glutathione in isolated selenoprotein W may suggest its involvement in the metabolism of this tripeptide.

Determination of two sites of automethylation in bovine erythrocyte protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase.

Protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed the alpha PCM fraction [Lindquist and McFadden (1994), J. Protein Chem. 13, 23-30]. The altered aspartyl sites serving as methyl acceptors in alpha PCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [3H]automethylated enzyme that contain a [3H]methyl ester. Methylation was positively identified at positions Asn188 and Asp217 in the enzyme sequence, a consequence of the spontaneous alteration of these sites to L-isoaspartyl or D-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows that alpha PCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.

Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation.

Oligomers possessing the Morpholino phosphorodiamidate backbone were evaluated for resistance to a variety of enzymes and biologic fluids. A 25-mer was incubated with nucleases, proteases, esterases, and serum, and the reaction mixtures were directly analyzed by MALDI-TOF mass spectrometry. The 25-mer was completely resistant to 13 different hydrolases and serum and plasma. The excellent resistance of Morpholino phosphorodiamidates to enzymatic attack indicates their suitability for in vivo use.

Alkylation of Escherichia coli thioredoxin by S-(2-chloroethyl)glutathione and identification of the adduct on the active site cysteine-32 by mass spectrometry.

Alkylation of reduced Escherichia coli thioredoxin by the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG) at physiologic pH resulted in at least three different alkylation products. These adducts were separated by reverse phase chromatography, digested with trypsin, and peptide-mapped. The peptide containing the active site cysteines was collected and sequenced by tandem mass spectrometry. Results indicate that the site of alkylation was at Cys-32 exclusively with no alkylation at Cys-35. Raising the pH above the pKa of Cys-35 to ionize the thiol before reacting with the episulfonium ion of CEG did not lead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic digest of a minor bis-adduct yielded an alkylated peptide which contained tyrosine, an amino acid known to be alkylated at its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsuccessful due to fragmentation of the alkylating moiety from the peptide. Results of this study confirm that the episulfonium ion of CEG can adduct thioredoxin at the active site and may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity.

Thioredoxin alkylation by a dihaloethane-glutathione conjugate.

Glutathione is a thiol-containing tripeptide which functions to protect cellular constituents from endogenous and xenobiotic electrophiles via conjugation and eventual excretion. In the case of compounds such as 1,2-dihaloethanes, however, conjugate formation results in bioactivation of the species rather than detoxification. The conjugate can then act as an alkylating agent toward cellular constituents including DNA, proteins, or lipids. Alkylation of protein thiols in cells exposed to dihaloethane may contribute substantially to the toxicity produced by these compounds. We examined the reactivity of the conjugate S-(2-chloroethyl)-glutathione (CEG) toward the model protein Escherichia coli thioredoxin. At physiological pH, treatment of thioredoxin by CEG resulted in the production of several bands visible on isoelectric focusing, which were determined by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to be mono-, di-, tri-, and tetra-alkylated forms of thioredoxin. A concomitant loss of in vitro enzymatic activity was observed. These products were also observed when reaction was allowed to take place at pH 11.4. Treatment at pH 4.4 resulted in lesser alkylation of thioredoxin, with only the mono- and di-alkylated forms detected. Iodoacetic acid treatment of CEG-alkylated thioredoxin revealed that the iodoacetic acid-susceptible Cys32 was not carboxymethylated, suggesting that this is one of the sites alkylated by CEG.

Purification and properties of selenoprotein W from rat muscle.

Following injection with [75Se]selenite, a low molecular weight 75Se-selenocysteine containing protein was purified from rat muscle. The purification procedure involved ammonium sulfate fractionation, Sephadex G-50 gel filtration, cation exchange chromatography on CM-Sephadex, and reverse phase high pressure liquid chromatography using a C-18 Vydac column. Four forms of the protein were separated by the cation exchange and reverse phase chromatography steps. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the four proteins revealed masses of 9550 +/- 1, 9596 +/- 1.2, 9858 +/- 1.3, and 9898 +/- 1.1 daltons. Glutamate, glycine, lysine, leucine, and valine are the major amino acids in this protein. About 0.92 g atoms of selenium was found per g mol of protein, and this selenium was present as selenocysteine. Thus, this appears to be a new selenoprotein, and we have named it selenoprotein W.

Sample derivatization and structure analysis by field desorption mass spectrometry. Peptide methylation-methanolysis.

Analysis by field desorption mass spectrometry of reaction product mixtures produced by treatment of peptides with methanol and hydrogen chloride augments significantly structural data derived by direct field desorption mass spectrometry analysis of the peptides. Analyses of data obtained by three peptides, beta-Ala-HisOH, p-Glu-Ser-GlyOH and Glu-Ser-Gly-AspOH are used for illustration. Data from field desorption mass spectrometry of peptide methylation-methanolysis product mixtures are used to (a) distinguish [M]+- from [M + H]+ in peptide field desorption mass spectrometry spectra, (b) ascertain the number of carboxyl groups present in a peptide, (c) identify a pyro-glutamyl N-terminus and (d) derive peptide sequence information. Definitive assignments of ion relationships are facilitated by use of methanol and deuteromethanol in paired experiments.