Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry.
Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.
Nikov
George
G
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Bhat
Vadiraja
V
Wishnok
John S
JS
Tannenbaum
Steven R
SR
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D015335:Molecular Probes; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis*
D015335:Molecular Probes; D011506:Proteins; D012709:Serum Albumin; C002744:3-nitrotyrosine; D014443:Tyrosine