Two-dimensional separation of the membrane protein sarcoplasmic reticulum Ca-ATPase for high-performance liquid chromatography-tandem mass spectrometry analysis of posttranslational protein modifications.

For the characterization of posttranslational modifications of the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA), we developed a two-dimensional separation protocol based on reversed-phase HPLC followed by SDS-PAGE and LC-MS/MS analysis of in-gel tryptic digests. Representative experiments are shown for the rabbit fast-twitch skeletal muscle isoform SERCA1. Matrix-assisted laser desorption-ionization and electrospray ionization-mass spectrometry analyses of SERCA1 tryptic digests revealed ca. 66% coverage of the protein sequence. This approach was used for the detection and quantitation of nitrotyrosine formation after exposure of SERCA1 to peroxynitrite in vitro. At molar ratios of nitrotyrosine to protein of 0.23 we confirmed by LC-MS/MS the nitration of predominantly Tyr(122) in the SERCA1 sequence.