Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line

Birgit Schilling; Srinivas Bharath M.M.; Richard H. Row; James Murray; Michael P. Cusack; Roderick A. Capaldi; Curt R. Freed; Kedar N. Prasad; Julie K. Andersen; Bradford W. Gibson

doi:10.1074/mcp.M400143-MCP200

Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line*S

From the ^‡Buck Institute for Age Research, Novato, CA 94945; ^¶Department of Molecular Biology, University of Oregon, Eugene, OR 97403; ^‖Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, and the Neuroscience Program, University of Colorado Health Sciences Center, Denver, CO 80262; ^**Center for Vitamins and Cancer Research, Department of Radiology, University of Colorado Health Sciences Center, Denver, CO 80262; ^‡‡Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143

§§ To whom correspondence should be addressed: Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945. Tel.: 415-209-2032; Fax: 415-209-2231; E-mail: bgibson{at}buckinstitute.org

Abstract

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson’s disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096–26101). Complex I is a ∼980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

Footnotes

Published, MCP Papers in Press, December 10, 2004, DOI 10.1074/mcp.M400143-MCP200
↵1 The abbreviations used are: PD, Parkinson’s disease; ETC, electron transport chain; SN, substantia nigra; ROS, reactive oxygen species; GSH, glutathione; IP, immunoprecipitation; mAb, monoclonal antibody; RNS, reactive nitrogen species; PTM, posttranslational modification; N27, rat dopaminergic neuronal cell line 1RB₃AN₂₇; PMF, peptide mass fingerprint; 1D, one dimensional; 2D, two dimensional; BN, Blue Native.
↵* This work was supported by National Institutes of Health Grant R21 NS043620-01 (to B. W. G.). S. B. M.M. is a recipient of a postdoctoral fellowship from the National Parkinson Foundation-Parkinson Disease Foundation Joint Program. The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material.
↵§ B. S. and S. B. M.M. contributed equally to this work.
- Received September 30, 2004.
- Revision received December 10, 2004.