DIFFERENTIAL S-NITROSYLATION OF PROTEINS IN ALZHEIMER’S DISEASE S. ZAHID, a,b R. KHAN, a M. OELLERICH, b N. AHMED a  * AND A. R. ASIF b  * a Neurochemistry Research Laboratory, Department of Biochemistry, University of Karachi, Karachi 75270, Pakistan b Department of Clinical Chemistry, University Medical Center Goettingen, Robert-Koch-Str. 40, 37075 Goettingen, Germany Abstract—Numerous studies have provided evidence regarding the involvement of protein S-nitrosylation in the progression of Alzheimer’s disease (AD) pathology and its implication in the formation and accumulation of misfolded protein aggregates. The identification of S-nitrosylated pro- teins can be a major step toward the understanding of mech- anisms leading to neuronal degeneration. The present study targeted S-nitrosylated proteins in AD hippocampus, substantia nigra and cortex using the following work-flow that combines S-nitrosothiol-specific antibody detection, classical biotin switch method labeled with fluorescence dye followed by electrospray ionization quadrupole time of flight tandem MS (ESI-QTOF MS/MS) identification. Endoge- nous nitrosocysteines were identified in 45 proteins, mainly involved in metabolism, signaling pathways, apoptosis and redox regulation as assigned by REACTOME and KEGG pathway database analysis. Superoxide dismutase (SOD2) [Mn], fructose-bisphosphate aldolase C (ALDOC) and volt- age-dependent anion-selective channel protein 2 (VDAC2) showed differential S-nitrosylation signal, not previously reported in AD regions. Extensive neuronal atrophy with increased protein S-nitrosylation in AD regions is also evi- dent from immunofluorescence studies using S-nitrosocys- teine antibody. A number of plausible cysteine modification sites were predicted via Group-based Prediction System-S- nitrosothiols (GPS-SNO) 1.0 while STRING 8.3 analysis revealed functional annotations in the modified proteins. The findings are helpful in characterization of functional abnormalities and may facilitate the understanding of molecular mechanisms and biological function of S-nitrosy- lation in AD pathology. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Alzheimer’s disease, proteomics, protein S-nitro- sylation, hippocampus, substantia nigra, cortex. INTRODUCTION S-nitrosylation, a result of the covalent binding of NO with cysteine residues of target proteins with the formation of nitrosothiols (SNOs), is a reversible post-translational modification. SNOs extensively modify protein function and play a key role in the pathology of multiple neurodegenerative diseases (NDDs) (Torta et al., 2008; Riederer et al., 2009; Nakamura et al., 2013). The dynamic and regulated balance of redox state of cysteine strongly affects the functional activity and interaction patterns of proteins as well as its localization and distribution within the cells (Ghafourifar et al., 2001). The reactive nitrogen species (RNS) may compromise cell function by protein modification through targeting tyrosine residues, thiols, and heme groups. Depending upon the severity of the cell damage RNS may also produce alterations in lipid oxidation pathways, damage DNA, inhibit mitochondrial respiration, and may also induce apoptosis and necrosis (Ricciardolo et al., 2004). In a number of cells (e.g., phagocytes, neurons, glial, endothelial and vascular cells) the production of reactive oxygen species (ROS), a consequence of increased nitric oxide (NO) due to expression of inducible NO synthase (iNOS or NOS2), may result in various deleterious effects including neurotoxicity and neuronal cell death (Boje, 2004). Increased expression of neuronal NOS (nNOS or NOS1) is observed in neurons with neurofibrillary tangles in the hippocampus and enthorinal cortex and in reactive astrocytes near amyloid plaques, while presence of neuritic plaques are also associated with increased expression of iNOS and endothelial NOS (eNOS or NOS3) in astrocytes (Calabrese et al., 2007; Knott and Bossy-Wetzel, 2009). Apoptotic cell death is a key cellular mechanism considered to be regulated by endogenous S- nitrosylation; depending upon the levels of cellular production of NO, where the low levels produce anti- apoptotic and elevated levels result in pro-apoptotic 0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.10.026 * Corresponding authors. Tel: +92-333-3880438 (N. Ahmed). Tel: +49-551-3922945 (A. R. Asif). E-mail addresses: nikhat_ahmed14@yahoo.co.uk (N. Ahmed), asif@med.uni-goettingen.de (A. R. Asif).   Equal contribution. Abbreviations: 2DE, two-dimensional electrophoresis; ACT, actin; AD, Alzheimer’s disease; ALDO, aldolase; BSM, biotin switch method; ENO, enolase; ESI-QTOF-MS/MS, electrospray ionization quadrupole time of flight tandem MS; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPS, group-based prediction system; HEPES, 2-[4- (2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; IP, immunoprecipi- tation; IPG, immobilized pH gradients; LDH, lactate dehydrogenase; NADP, nicotinamide adenine dinucleotide phosphate; NDDs, neurodegenerative diseases; NEM, N-ethylmaleimide; NO, nitric oxide; PEBP, phosphatidylethanolamine-binding protein; PTMs, post- translational modifications; RNS, reactive nitrogen species; SNO, S- nitrosothiols; SNO-Cys, S-nitrosocysteine; SOD2, superoxide dismutase; TBST, Tris-buffered saline and Tween 20; TPI, triose phosphate isomerase; TUBA, tubulin alpha; TUBB, tubulin beta; VDAC2, voltage-dependent anion-selective channel protein 2; WEBGESTALT, WEB-based Gene SeT AnaLysis Toolkit. Neuroscience 256 (2014) 126–136 126