T2 PM 'Omic' Technologies: Current and Future Applications to Environmental Toxicology (Part 2)
Tuesday, 15 November 2005: 1:50 PM - 5:30 PM in Ballroom 2

(TRI-1117-847015) Probing Molecular Mechanisms of Axonal Neuropathy by a Functional Proteomics Approach.

Trimpin, S^{1, 2}, Deinzer, M², Spencer, P¹, Cranson, A¹, Hashemi, S^{1, 3}, Lasarev, M¹, Pounds, J³, Palmer, V¹, Sabri, M¹, Tshala-Katumbay, D¹, ¹ Center for Research on Occupational & Environ. Toxicol., Oregon Health & Science University, Portland, OR, USA² Oregon State University, Corvallis, OR, USA³ Pacific Northwest National Laboratory, Richland, WA, USA

ABSTRACT- Background: Certain aromatic (1,2-diethylbenzene) and aliphatic solvents (n-hexane) - the latter causing occupational neuropathy in China and elsewhere - rapidly form protein-reactive gamma diketone metabolites that induce murine neurofilamentous axonopathy in proximal (intraspinal and spinal roots) and distal (nerve) regions of large-diameter myelinated fibers, respectively. Neuroprotein vulnerability in vitro and in vivo correlated directly with lysine content, which formed cross-linked polymers selectively with 1,2-DAB. Analysis of brain cytosol by cLC-FTICR-MS revealed over 800 proteins: several brain proteins were markedly increased in 1,2-DAB vs. 1,3-DAB/Vehicle (1 week), including the microtubule-dissociating protein stathmin. This lysine-rich protein is of great interest because elderly mice lacking the gene (sthm-1) for stathmin spontaneously develop axonopathy [Liedtke et al., Am. J. Pathol. 160:469, 2002]. Methods: A mini-ball mill (MBM) sample preparation procedure was developed for solvent-free MALDI analysis of peptides and proteins [Trimpin, S.; Deinzer, M. L. J. Amer. Mass Spectrom. 16: 542, 2005.]. MBM MALDI-MS was compared to conventional solvent-based MALDI-MS towards the analysis of H/D-ratio measurements from exchange experiments (e.g., MW determination and peptide mapping investigations of stathmin prior and after H/D-exchange experiments) to secure sufficient retention of label to ensure accurate measurements applying the MALDI-TOF/TOF high energy collision fragmentation, hence, measurements that show undesired proton back-exchange in conventional MALDI and ESI-MS approaches also due to solvent-sensitivity. Results: A solvent-free MBM MALDI-MS method was developed that appears to be an improvement over standard solvent-based MS procedures for the determination of H/D-ratio measurements due to reduced back-exchange and a promising extension to global proteomics studies. Conclusion: The foregoing strategy, global proteomics followed by a more in-depth characterization (protein conformation) of a target protein of interest (stathmin), holds promise for the identification of cellular protein networks and changes upon chemically induced stress in the molecular pathogenesis of axonal neuropathy [Supported by NIEHS grants ES11384, ES10338].

Key words: functional proteomics, biomarker, stathmin, H/D-exchange