Quantitation of N epsilon-(dichloroacetyl)-L-lysine in proteins after perchloroethene exposure by gas chromatography-mass spectrometry using chemical ionization and negative ion detection followingimmunoaffinity chromatography.
An antibody specific to N epsilon-(dichloroacetyl)-L-lysine (DCA-Lys) was immobilized to immunoaffinity columns for the use in selective enrichment of dichloroacetylated proteins. These result from the reaction with dichlorothioketene the beta-lyase cleavage product of the perchloroethene metabolite S-(trichlorovinyl)-L-cysteine. Dichloroacetylated proteins from rat kidney mitochondria, rat plasma and human blood plasma were isolated after exposure to 40 ppm tetrachloroethene (PER) for 6 h. After acid hydrolysis of the protein fraction, DCA-Lys was derivatized with 1,3-dichloro-1,1,3,3-tetrafluoroacetone using N epsilon-(trifluoroacetyl)-L-lysine as internal standard. Recovery of dichloroacetylated reference proteins from immunoaffinity columns was about 73%. Samples were analyzed by GC-MS with chemical ionization and negative ion (NCI) detection showing DCA-Lys in proteins with 2.26 (+/- 0.02) pmol/mg protein in male rat kidney mitochondria and 1.92 (+/- 0.05) pmol/mg total mitochondrial protein in female rats. In rat plasma 0.47 (+/- 0.006) pmol DCA-Lys/mg protein in male and 0.34 (+/- 0.02) in female animals were found. DCA-Lys could not be detected in blood plasma of human volunteers exposed to PER with a detection limit of 20 fmol for the DCA-Lys derivative 2,2-bis(chlorodifluoromethyl)-4-(1-dichloroacetamido)-butyl- 1,3-oxazolidine-5-one. Immunoaffinity chromatography with specific antibodies provides a powerful tool for the enrichment of minor quantities of dichloroacetyled proteins in biological samples for GC-NCI-MS analysis of the modified amino acid lysine having broad utility in the biomonitoring of PER exposure.
Pähler
A
A
Institut für Toxikologie, Universität Würzburg, Germany.
Völkel
W
W
Dekant
W
W
D000328:Adult; D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis; D008297:Male; D008875:Middle Aged; D016273:Occupational Exposure; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D013750:Tetrachloroethylene / Q000633:toxicity*
D011506:Proteins; C092783:N(epsilon)-(dichloroacetyl)lysine; D008239:Lysine; D013750:Tetrachloroethylene