Figure 6.

Mass spectrometric identification of eNOS phosphorylation at Ser¹¹⁷⁶ in rat hearts subjected to IPC. (A) MS/MS spectrum of doubly protonated molecular ion, m/z = 588.2649²⁺ of amino acids 1174–1182 of the eNOS C-terminal region. Rat eNOS was purified from IPC hearts by immunoprecipitation with eNOS antibody. The phosphorylation site of eNOS at Ser¹¹⁷⁶ in rat heart was determined from tryptic fragment ₁₁₇₄TQS_(PO4)¹¹⁷⁶FSLQER₁₁₈₂. MW difference of 80 Da is seen between fragment ions y6 and y7 compared with the native fragment ions, which confirmed phosphorylation of Ser¹¹⁷⁶. The sequence-specific ions were labelled as y and b ions. (B) Molecular model of the eNOS reductase site with phosphorylation at Ser¹¹⁷⁶ showing its critical position in the C-terminal α-helix close to the critical FAD cofactor.