Project PXD000746

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Biomedical Dataset
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Summary

Title

Proteomics of Fuchs Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet¹s membrane is Disordered

Description

Fuchs’ endothelial corneal dystrophy is major corneal disorder in the western world affecting the innermost part of the cornea, which leads to visual impairment. The morphological changes observed in Fuchs’ endothelial corneal dystrophy is well described, however, much less in known of the pathology at the molecular level. As the morphological changes observed in the cornea is profound in the extracellular matrix we sought to determine in protein profiles and changes herein in the Descement’s membrane and endothelium layer of Fuchs’ endothelial conrneal dystrophy patients when compared to healthy control tissue. Using the extracted ion chromatogram label-free MS based quantification method we quantified approximately the 50 most abundant proteins of the Descemet’s membrane and endothelial layer in in patient and control tissue. In addition, using the isobaric tag for relative and absolute quantification MS method resulted in a total of 22 regulated proteins of which the majority were extracellular proteins known to be involved in proper assembly and modulation of the basement membrane in other tissues. Many of the regulated proteins were furthermore among the most abundant proteins quantified. The two MS methods performed here suggest altered arrangement of the extracellular matrix in Fuchs’ endothelial corneal dystrophy and provide new candidate proteins that may be involved in molecular mechanism of this disease.

Sample Processing Protocol

Sample preparation for LC-MS/MS – in-solution digest – Patient and control tissues were lyophilized for 10 h using a speedvac concentrator before subjection to 0.66 M CNBr in 70% trifluoroacetic acid overnight at 23°C. Then, tissues were lyophilized and re-suspended in 8 M Urea in 0.2 M Tris-HCl, pH 8.3, reduced in 15 mM dithiothreitol for 1 h, and alkylated in 30 mM iodoacetamide for 1 h. The samples were then diluted 5 times with 0.1 M Tris-HCl, pH 8.3, and digested overnight with 1:50 w/w sequencing grade modified trypsin (Promega, Madison, WI, USA) at 37°C for 16 h in a final volume of 0.5 mL. The samples were desalted using POROS 50 R2 RP column material (Applied Biosystems, Foster City, CA, USA) packed in GELoader Tips (Eppendorf, Hamburg, Germany). iTRAQ labeling and strong cation-exchange fractionation – Isobaric tag for relative and absolute quantification (iTRAQ) analysis was performed using 10 µg of in-solution digested sample from each patient and control. Samples were labeled with iTRAQ 4plex (AB SCIEX, Framingham, MA, USA) according to the manufacturer’s protocol for 1 hour and mixed according to figure 1, resulting in a total of five sets of 4plex experiments. A mixed sample containing 5 µg sample from each of the patients and controls was labeled with the iTRAQ 114-tag in all five experiments and used to normalize between experiments. Each 4plex experiment was diluted 10 times in buffer A (0.5% formic acid and 5% acetonitrile) and separated by strong cation exchange (SCX) using a PL-SCX 1000 Å 5 μm 20 × 2.1 mm column (Higgins Analytical, Rengstorff, CA, USA) equilibrated in buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa, WI, USA) at a flow rate of 100 μL/min. The peptides were eluted using a 1% B/min linear gradient from buffer A to buffer B (0.5% formic acid, 5% acetonitrile containing 1 M NaCl) into 15 fractions. The samples were lyophilized and desalted using POROS 50 R2 material packed in GELoader tips. The desalted samples were lyophilized and resuspended in 12 μL 0.1% formic acid and stored at −20 °C. LC-MS/MS analysis – LC-MS/MS analyses were performed on an EASY-nLC II system (Thermo Fisher Scientific, Waltham, MA, USA) connected to a TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with a NanoSpray III source (AB SCIEX, Framingham, MA, USA) and operated under Analyst TF 1.6.0 control. The CNBr and trypsin cleaved samples were dissolved in 0.1% formic acid, injected and trapped on a Biosphere C18 column (5 μm, 2 cm x 100 μm I.D; Nano Separations, Nieuwkoop, Netherlands). Next, the peptides were eluted from the trap column and separated on a 15 cm analytical column (75 μm i.d.) packed in-house in a fritted silica tip (New Objectives, Woburn, MA, USA) with RP ReproSil-Pur C18-AQ 3 μm resin (Dr. Marisch GmbH, Ammerbuch-Entringen, Germany) connected in-line to the mass spectrometer. Peptides were eluted at a flow rate of 250 nL/min using a 50 min gradient from 5% to 35% phase B (0.1% formic acid and 90% acetonitrile). The acquisition method used for the extracted ion chromatogram (XIC) quantification was set up as a information-dependent acquisition (IDA) with 25 MS/MS spectra per cycle and a 1.6 sec cycle times using an exclusion window of 6 sec. For iTRAQ experiments the IDA settings were changed to 50 MS/MS per cycle using a 2.8 sec cycle time. Two iTRAQ experiments were performed on each 4plex set either with iTRAQ collision energy adjustment enabled or disabled.

Data Processing Protocol

XIC quantitation – All MS files were processed using Skyline v. 2.1.0.4936 (MacCoss Lab, University of Washington). The MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.3 (AB SCIEX) and the "proteinpilot MGF" parameters. The peak lists were used to interrogate the Swiss-Prot (v. 2013_11, 541,762 sequences) Homo sapiens (20,279 sequences) database using Mascot 2.3.02. Trypsin was employed as enzyme allowing one missed cleavage. Carbamidomethyl was entered as a fixed modification, whereas oxidation of methionine and hydrozylation of proline were entered as variable modifications. The mass accuracy of the precursor and product ions were 10 ppm and 0.2 da, and the instrument setting was specified as ESI-QUAD-TOF. The significance threshold (p) was set at 0.01 and the ion score expect cut-off at 0.005. Mascot dat result files were used to generate a spectral library in Skyline using the human sequences from Swiss-Prot as background proteome. Employing the same parameters as for the Mascot search, the three most abundant peptides for each protein were manually chosen from all peptides available in the spectral library. Proteins identified with less than 3 peptides were not included in the XIC assay. In addition, only peptides quantified with an idotp value≥ 90 were included for XIC quantification. This label-free quantification protocol relies on the average MS signal response for the three most intense tryptic peptides for each protein. The relative abundance and standard deviation of proteins quantified in a minimum of three of the technical replicates was calculated as the average MS intensity for the three peptides for each protein divided by the sum of the average signal for all quantified proteins in the sample. A student’s t-test (p=0.05) was applied to test the XIC data for significant regulated proteins between the FECD and control group. The student’s t-test and average protein amounts shown in table 1 was only calculated on proteins quantified in a minimum of six patients in the FECD group and a minimum of three individuals in the control group. iTRAQ quantification – The collected iTRAQ MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.3 (AB SCIEX, Framingham, MA, USA) and the "proteinpilot MGF" parameters. For each 4plex set the peak lists for the two iTRAQ experiments with different collision energy settings were merged and used to interrogate the Swiss-Prot (version 2013_11) Homo sapiens (20,279 sequences) database using Mascot 2.3.02 (Matrix Science, Boston, MA, USA). Trypsin was employed as enzyme allowing one missed cleavage. Carbamidomethyl was entered as a fixed modification, hydroxylation of proline was entered as a variable modification and all methionine-containing peptides were neglected. The mass accuracy of the precursor and product ions were 10 ppm and 0.2 dalton, the instrument setting was specified as ESI-QUAD-TOF, and the iTRAQ 4plex protocol was selected as quantitation method. The significance threshold (p) was set at 0.01, the ion score expect cut-off at 0.005, and all iTRAQ rations were normalized based on summed intensities. Mascot iTRAQ results were parsed using MS Data Miner v. 1.2.2 and only proteins quantified with a minimum of 3 unique peptides were included for further analysis. To find significantly regulated proteins, a student’s t-test with a significant threshold of 0.05 was performed for all proteins quantified in more than half of the biological replicates from each group (FECD and Controls) (Supplementary Table 2).

Contact

Ebbe Toftgaard Poulsen, Department of Molecular Biology and Genetics
Jan Johannes Enghild, Department of Molecular Biology and Genetics, Aarhus University, Denmark ( lab head )

Submission Date

07/02/2014

Publication Date

27/05/2014

Publication

    Poulsen ET, Dyrlund TF, Runager K, Scavenius C, Krogager TP, Hojrup P, Thøgersen IB, Sanggaard KW, Vorum H, Hjortdal J, Enghild JJ. Proteomics of Fuchs' Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet's membrane is disordered. J Proteome Res. 2014 May 21 PubMed: 24846694

Assay

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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 34732 iTRAQ set 1 222 23329 977 1009562 22616
2 34731 iTRAQ set 1 266 20661 1192 1092099 19805
3 34734 iTRAQ set 3 280 19375 1215 996355 18228
4 34733 iTRAQ set 4 338 28463 1418 1004816 27198
5 34730 iTRAQ set 5 289 15380 1311 997695 14364
6 34739 XIC quantification 132 1978 661 29384 1911
7 34736 XIC quantification 126 1976 626 29019 1900
8 34735 XIC quantification 120 1958 620 28905 1901
9 34738 XIC quantification 126 1970 642 29556 1906
10 34737 XIC quantification 121 1912 635 28568 1824