1. PROTOCOL OPTIMIZATION
CHARACTERIZING THE PROTEIC DEPOSIT IN FELINE AMYLOIDOSIS: A PROTEOMIC APPROACH USING FFPEs
Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Via Celoria 10- Milano, Italy;
Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO USA
Francesca Genova1, Maria Longeri1, Leslie A. Lyons 2, Francesca Grassi Scalvini1, Gabriella Tedeschi1, Giuseppe Sironi1, Simona Nonnis1
BACKGROUND
Amyloidosis is a group of primary and secondary diseases characterized by the abnormal deposition of insoluble fibrillary proteins in different organs . Mammals, including felids and humans, are affected. More than twenty-eight types of Amyloidosis have been identified in humans occurring via genetic mutations (hereditary), proteins increased production or decreased elimination, and misfolding disorders (in Alzheimer). In cats Amyloidosis is diagnosed with certainty only by post-mortem identification of the deposits. Etiology is quite obscure and the fine characterization of the protein deposits is not available yet. Preliminary results obtained in our laboratory from the proteomic analysis of few affected and normal frozen tissues of Abyssinian cats, suggest that the expression of some proteins is regulated in Amyloid deposits.
AIM
The aim of this project is to characterize amyloid deposits in Felis catus kidneys by a mass spectrometric proteomic approach. We started setting up a protocol for frozen tissues (data not shown) but, since it is difficult to obtain fresh tissues from affected cats, we optimized a new protocol for Formalin Fixed Paraffin Embedded (FFPE), which are available in the university archives.
PROTOCOL OPTIMIZATION
Ten archived kidney FFPE blocks, from 5 affected and 5 healthy Abyssinian cats, were used for the protocol optimization. Five unstained standard histological slices from each block have been prepared (Figure1).
The protein components obtained were reduced and alkylated prior to the triptic digestion (protein:enzyme 20:1) overnight at 37°C. Acidification with 0.3% TFA blocked the digestion reaction and peptide mixures ware desalted using Zip-Tip C18 before mass spectrometric analysis.
Peptides were loaded directly on an HPLC system coupled online to an LTQ-Orbitrap Velos mass spectrometer. Data acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software (Thermo Fisher Scientific). The LTQ-Orbitrap was operated in positive mode in data-dependent acquisition mode to automatically alternate between a full scan (m/z 350–2000) in the Orbitrap (at resolution, 60,000; AGC target, 1,000,000) and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan (normalized collision energy of 35%, 10-ms activation) (Figure2). Data Base searching was performed using the Sequest search engine contained in the Proteome Discoverer 1.4 software against Felis catus database. The following parameters were used: 10 ppm for MS and 0.5 Da for MS/MS tolerance, carbamidomethylation of Cys as fixed modification and oxidation (M), deamidation (N,Q) and acetylation (N-term) as variable modifications, trypsin (two misses) as protease. To generate the list of proteins reported in Table 3, we considered only the peptides with the highest X Correlation value (Xcorr) in Seques (>1.5) and the best fragmentation pattern, selected manually after visual inspection of the MS/MS spectra (Figure3).
Figure 1: Workflow
Figure 3: Representative MS/MS spectrum
RESULTS
One technical replicate of each sample was analyzed leading to the identification and quantification of protein groups in FFPEs preparations of healthy and amyloid specimens. Raw file analysis, performed using Proteome Discoverer 1.4 software, allowed to identify proteins related to amyloidosis in cats (Table1).
Figure 2: Schematic overview of MS sample preparation and data analysis
Table 1: Identified amyloid fibril proteins
CONCLUSIONS
Interestingly, proteins which were previously characterized in amyloid deposits on frozen tissues, are recognized to form deposits in amyloid kidneys in FFPEs. The identification of these proteins provides new information to understand molecular mechanisms of familial amyloidosis in cats which have been also inquired by genomic approach (see Poster n. MT103)
THIS WORK IS SUPPORTED BY LINEA 2 “PIANO DI SOSTEGNO PER LA RICERCA ” UNIMI