Proteomic strategies for identifying protein modifications and their structural changes

Abstract

Reactive oxygen species (ROS) is known as a second messenger in non-phargocytic cells. However, the molecular mechanisms of ROS action are not well understood. In this study, cutting edge proteomic tools have been developed and applied in order to identify the target proteins of ROS, modified species/sites of ROS target proteins and the structural changes by oxidative modifications. Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, are known to regulate the biological function of various proteins. This study has identified novel low abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing new strategy of selectively excluded mass screening analysis (SEMSA) for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight (nanoUPLC-ESI-q-TOF) tandem mass spectrometry (MS), in conjunction with newly developed MODi and MODmap algorithm. Unexpected mass shifts (△m = -16, -34, +64, +87, and +103 Da) at redox-active cysteine residue were observed in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, model compounds were prepared and the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (m/z -34 Da) or serine along with Cys-SO2-SH (△m = +64 Da) examined. Also unexpected acrylamide adducts of sulfenic and sulfinic acids (△m = +87 and +103 Da) were detected. These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine. To investigate the structure changes of redox sensitive proteins in response to ROS, Nm23-H1/NDPK-A, a tumour metastasis suppressor, was employed as a model system. Nm23-H1 is a multifunctional housekeeping enzyme with nucleoside diphosphate kinase activity, and its hexameric form is required for suppression of tumour metastasis and is readily dissociated into dimers under oxidative conditions. Here, the crystal structure of oxidized Nm23-H1 is presented. It reveals the formation of an intramolecular disulfide bond between Cys4 and Cys145 that triggers a large conformational change that destabilizes the hexameric state. The dependence of the dissociation dynamics on the H2O2 concentration was determined using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) methodology. The quaternary conformational change provides a suitable environment for the oxidation of Cys109 to sulfonic acid, as demonstrated by peptide sequencing using nanoUPLC-ESI-q-TOF tandem MS. From these and other data, it is proposed that the molecular and cellular functions of Nm23-H1 are regulated by a series of oxidative modifications coupled to its oligomeric states and that the modified cysteines are resolvable by NADPH-dependent reduction systems. These findings broaden the understanding of the complicated enzyme regulatory mechanisms that operate under oxidative conditions. For the understanding the comprehensive disulfide formation and their localization of oxidized proteins, quantitative analysis of oxidized proteins in response to H2O2 were perfomed combining cellular fractionation into cytosol, plasma membraned, nucleus and nuclear membrane, iTRAQ labeling and identification with tandem MS. Results demonstrate that in-gel based iTRAQ coupled DBond algorithm facilitated comprehensive identification of redox reactive proteins and their localization changes under the non-reducin...