Figure 2.

Confirmation of apoA-I as a nitrated protein by both 2D SDS-PAGE and anti-NO₂Tyr affinity chromatography coupled to tandem MS-based sequencing. Plasma from a subject with CVD was loaded onto an affinity matrix composed of immobilized affinity-purified rabbit anti-NO₂Tyr polyclonal antibodies, washed with high salt, and then eluted with addition of 5 mM free NO₂Tyr, as described in Methods. (A) Demonstration of apoA-I location on 2D SDS-PAGE. Identity of protein was established by tandem MS sequence analysis of peptides (>95% coverage). (B) The anti-NO₂Tyr eluent (5 mM NO₂Tyr) was subjected to 2D SDS-PAGE, and the presence of apoA-I was confirmed by Western blot analysis. Parallel studies using control nonimmune IgG as the affinity matrix failed to bind detectable levels of apoA-I (not shown).