title: Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC-electrospray-tandem MS: selective protein oxidation during biological aging.
abstract: The selective reversible S-glutathiolation of specific SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) cysteine residues represents a novel physiologic pathway of NO (nitric oxide)-dependent arterial smooth muscle relaxation [Adachi, Weisbrod, Pimentel, Ying, Sharov, Schöneich and Cohen (2004) Nat. Med. 10, 1200-1207]. This mechanism may be impaired through the irreversible oxidation of functionally important cysteine residues as a consequence of oxidative stress and aging. To establish whether in vivo aging and in vitro oxidation by peroxynitrite result in the loss of such functionally important cysteine residues of SERCA, we have developed and optimized a quantitative method to monitor the oxidation state of the individual SERCA cysteine residues using a maleimide-based fluorescence dye, TG1 (ThioGlo 1), as a label for cysteine residues that have not been altered by oxidation and are not involved in disulphide bridges. A high efficiency for TG1 labelling of such residues and the chemical structure of cysteine-TG1 adducts were validated by MS analysis of model peptides, model proteins and rat skeletal muscle SERCA1. Tryptic peptides containing 18 out of a total of 24 cysteine residues were identified by HPLC-ESI (electrospray ionization)-MS/MS (tandem MS). Two cysteine residues, at positions 344 and 349, were detected in the form of an internal disulphide bridge, and another 16 were found to be labelled with TG1. Using HPLC-ESI-MS, we quantitatively mapped peroxynitrite oxidation of eight cysteine residues (positions 364, 417, 420, 498, 525, 674, 675 and 938), some of which are involved in the control of SERCA activity. Biological aging resulted in the partial modification of cysteine residues 377, 498, 525, 561, 614, 636, 674, 675, 774 and 938. Neither peroxynitrite exposure nor biological aging affected the apparent SERCA1 ATP affinity. Our data show an age-dependent loss of cysteine residues (approx. 2.8 mol of cysteine/mol of SERCA1), which may be partially responsible for the age-dependent decrease in the specific Ca2+-ATPase activity (by 40%).
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
authors.forename: Victor S
authors.initials: VS
authors.lastname: Sharov
authors.forename: Elena S
authors.initials: ES
authors.lastname: Dremina
authors.forename: Nadezhda A
authors.initials: NA
authors.lastname: Galeva
authors.forename: Todd D
authors.initials: TD
authors.lastname: Williams
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
mesh_terms: D000375:Aging / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes; D010084:Oxidation-Reduction; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: C506945:Atp2a1 protein, rat; D005456:Fluorescent Dyes; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003545:Cysteine