Improved tandem mass spectrometric characterization of 3-nitrotyrosine sites in peptides.
Nitrated tyrosines are easily converted into their aminotyrosine equivalents by a reduction step. We here show that this conversion can be exploited to readily discern 3-aminotyrosine peptides in a background of non-nitrated peptides. Furthermore, aminotyrosine peptides are more stable in single mass spectrometry (MS) mode rendering peptide mass maps easier to interpret. One significant caveat of both 3-nitrotyrosine and 3-aminotyrosine peptides is their lack of efficient fragmentation upon collision-induced dissociation (CID) which, in the case of the latter peptides, also produces unexpected, deviating isotopic patterns of fragment ions containing the aminotyrosine residue. The net result is that sequence database searching becomes daunting as the correct peptide is frequently missed since insufficient and/or inaccurate peptide fragments are used. We show that a simple acetylation step, blocking all amines (including aminotyrosine), produces peptides that undergo extensive backbone fragmentation by CID and are thus easily identifiable in databases. Our procedure is additionally illustrated by doubling the number of nitration events mapped in tetranitromethane-nitrated bovine serum albumin (BSA) as compared to a direct analysis of the nitrated peptides using the same amount of material. In conclusion, we here illustrate that this two-step process, heme-mediated reduction and acetylation, can be used for more efficient characterization of protein-bound nitrated tyrosines.
Ghesquière
Bart
B
Department of Medical Protein Research and Biochemistry, Flanders Interuniversity Institute for Biotechnology and Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.
Goethals
Marc
M
Van Damme
Jozef
J
Staes
An
A
Timmerman
Evy
E
Vandekerckhove
Joël
J
Gevaert
Kris
K
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D010455:Peptides; D012710:Serum Albumin, Bovine; C002744:3-nitrotyrosine; D014443:Tyrosine