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.

Characterising the Mechanism of Airway Smooth Muscle β2 Adrenoceptor Desensitization by Rhinovirus Infected Bronchial Epithelial Cells

“…In order to identify whether there was a difference in the peptide profiles between the control and RV conditioned medium that were in the <3 kDa fraction, mass spectrometry (MS) was carried out as previously described by Ly et al [28]. In brief, the samples were purified using Ziptips (Millipore) as per the manufacturer’s instructions.…”

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

“…Specifically, the DT workflow entails an initial tissue homogenization, which can be performed using different buffers, such as the commercial Liquid Tissue [16], as well as mixtures of ammonium bicarbonate (ABC) and acetonitrile (ACN) [17,18] or ABC and trifluoroethanol [19,20], followed by direct trypsin digestion of the tissue homogenate. The ISD workflow is instead based on a preliminary protein extraction step, comprising tissue lysis in a detergent-based buffer (usually containing SDS) and collection of soluble proteins after centrifugation, succeeded by detergent depletion (by means of dilution with ABC [21,22], dialysis [23], spin columns [24] or protein precipitation with various protocols [25-28]) and in solution digestion of the protein extract. The FASP protocol, applied with remarkable results to FFPE samples by Wiśniewski and colleagues [29-32] and recently employed with modifications by other research groups [33,34], shares the initial protein extraction step with the ISD workflow but differs in that detergent removal and protein digestion are both performed on a molecular weight cut-off centrifugal filter [35,36], instead of in solution.…”

Discovery of Novel Disease-specific and Membrane-associated Candidate Markers in a Mouse Model of Multiple Sclerosis

“…Differential gene and protein expression profiles have been generated based on comparative analyses of healthy control and disease-affected tissues derived from clinical samples (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and animal models (19 -29). These biomarker discovery platforms include gel-based approaches such as two-dimensional gel electrophoresis (2D-GE) (10,17,30), 2D-difference image gel electrophoresis (2D-DIGE) (9,14), as well as shotgun proteomics techniques (11,13,16,31,32) incorporating the use of label-free or stable isotope labeling LC-MS-based strategies for quantitative proteomic studies.…”