A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: enzymatic digestion versus hydrochloric acid-mediated hydrolysis.
A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.
Delatour
Thierry
T
Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland. thierry.delatour@rdls.nestle.com
Fenaille
François
F
Parisod
Véronique
V
Richoz
Janique
J
Vuichoud
Jacques
J
Mottier
Pascal
P
Buetler
Timo
T
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D006851:Hydrochloric Acid / Q000378:metabolism*; D006868:Hydrolysis; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D004798:Enzymes; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine; D006851:Hydrochloric Acid