title: Comprehensive tissue processing strategy for quantitative proteomics of formalin-fixed multiple sclerosis lesions.
abstract: Formalin-fixed (FF) autopsy tissue comprises the bulk of existing Multiple Sclerosis (MSc) pathology archives, providing a rich pool of material for biomarker discovery and disease characterization. Here, we present the development of a heat-induced extraction protocol for the proteomic analysis of FF brain tissue, its application to the study of lesion remyelination and its failure in MSc. A 4-round extraction strategy was optimized using FF tissue leading to a 35% increase in the number of proteins identified compared to a single extraction; and a 65% increase in proteins identified with ≥4 peptides. Histological staining of sections with oil red O and luxol fast blue-periodic acid Schiff, required to characterize MSc lesions was found to have minimal effect on LC-MS/MS. The application of the optimized protocol to chronic demyelinated and remyelinated FF MSc lesions and the adjacent periplaque white matter, isolated through laser guided manual dissection from 3 patients, identified 428 unique proteins (0.2% FDR) using LC-MS/MS. Comparison of the lesion types using iTRAQ and 2-D LC-MS/MS revealed 82 differentially expressed proteins. Protein quantitation by iTRAQ and spectral counting was well-correlated (r(s)= 0.7653; p < 10(-30)). The data generated from this work illustrates the scope of the methodology and provides insights into the pathogenesis of MSc and remyelination.
authors.affiliation: Central Clinical School, The University of Sydney, NSW, Australia.
authors.forename: Linda
authors.initials: L
authors.lastname: Ly
authors.forename: Michael H
authors.initials: MH
authors.lastname: Barnett
authors.forename: Yuan Z
authors.initials: YZ
authors.lastname: Zheng
authors.forename: Twishi
authors.initials: T
authors.lastname: Gulati
authors.forename: John W
authors.initials: JW
authors.lastname: Prineas
authors.forename: Ben
authors.initials: B
authors.lastname: Crossett
mesh_terms: D000328:Adult; D000368:Aged; D001344:Autopsy; D001391:Azo Compounds / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D007249:Inflammation; D007554:Isotopes / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000378:metabolism*; D009186:Myelin Sheath / Q000737:chemistry; D010504:Periodic Acid / Q000494:pharmacology; D040901:Proteomics / Q000379:methods*
chemical_list: D001391:Azo Compounds; D007554:Isotopes; D010504:Periodic Acid; C011049:oil red O