Proteomic method for identification of tyrosine-nitrated proteins.
Biologic nitration of protein tyrosine (to form 3-nitrotyrosine) is a recently described phenomenon that is associated with many diseases. We have devised a proteomic methodology to identify these modified proteins. This utilizes protein fractionation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), partial transfer onto polyvinylidene difluoride (PVDF) membranes, and Western blot analysis using an antinitrotyrosine antibody to identify the proteins. Alignment of the Western blots with the partially transferred 2-D PAGE gels enables identification of immunopositive protein spots. These are then excised and trypsin digested. Proteins are then identified using either matrix-assisted laser desorption ionization-time of flight mass spectrometry or capillary liquid chromatography tandem electrospray mass spectrometry. Nonspecific crossreactivity of the antibodies is determined using reduction of protein bound 3-nitrotyrosine to 3-amino tyrosine using sodium dithionite.
Aulak
Kulwant S
KS
Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, OH, USA.
Koeck
Thomas
T
Crabb
John W
JW
Stuehr
Dennis J
DJ
D000818:Animals; D002478:Cells, Cultured; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
D011506:Proteins; D019000:Phosphotyrosine; C002744:3-nitrotyrosine; D014443:Tyrosine; D014357:Trypsin