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PXD000307

DataSet Summary

  • HostingRepository: PRIDE
  • AnnounceDate: 2014-07-28
  • AnnouncementXML: Submission_2014-07-28_00:40:10.xml
  • DigitalObjectIdentifier: http://dx.doi.org/10.6019/PXD000307
  • ReviewLevel: Peer-reviewed dataset
  • DatasetOrigin: Original data
  • RepositorySupport: Supported dataset by repository
  • PrimarySubmitter: Ebbe Toftgaard Poulsen
  • Title: Comparison of phenotypically distinct lattice corneal dystrophies reveals similar plaque proteomes and suggests altered proteolytic turnover rates of the transforming growth factor beta-induced protein (TGFBIp)
  • Description: *Abstract:* In this study, we investigated whether the phenotypic difference observed between the two siblings with an atypical variant of lattice corneal dystrophy type 1 (LCD type 1) caused by a single A546D substitution and a A546D/P551Q double substitution in TGFBIp, can be ascribed to (i), a difference in plaque proteomes (ii), altered proteolysis of TGFBIp or (iii), is a consequence of the P551Q amino acid substitution. Amyloid deposits were isolated from two corneal specimens with atypical LCD type 1 due to a A546D/P551Q mutation in TGFBI using laser capture microdissection and a subsequent analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The findings were compared to the previous findings of a A546D LCD type 1 plaque proteome. Proteolytic processing of TGFBIp was addressed by counting peptide spectra obtained during the mass spectrometry (MS) analyses. Lastly, to study the effect of the P551Q substitution, recombinant FAS1-4 variants (wild type, A546D, A546D/P551Q, and P551Q) were expressed, purified and subjected to in vitro stability assays including circular dichroism, transverse urea gradient gel electrophoresis, and limited proteolysis. Plaque proteomes of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to A564D plaque proteome previously reported. Major proteins associated with the deposits were TGFBIp, serum amyloid P, clusterin, HtrA1, Apolipoproteins A-1, A-IV, D, and E. Proteolytic processing of TGFBIp were also similar in all LCD type 1 cases regardless of the mutation indicating that a C-terminal TGFBIp polypeptide had accumulated in the deposits. The stability assays revealed a minor destabilization of FAS1-4 upon the addition of the P551Q mutation, however, enough to result in different accessibility to proteolytic processing by trypsin for the A546D and A546D/P551Q FAS1-4 variants. Difference in A546D and A546D/P551Q LCD type 1 phenotypes is not ascribed to altered plaque formation or altered proteolytic processing of TGFBIp. Instead, a small decrease in thermodynamic stability introduced by the P551Q mutation is believed to affect TGFBIp fibrillation and proteolytic accessibility of TGFBIp, thereby resulting in the different phenotypes observed in vivo. *Data handling:* Raw data were converted to mgf format using Protein Pilot software (AB Sciex) and searched using an in-house Mascot search engine (Matrix Science, London, UK; version: 2.3.02) against the Swiss-Prot database (version: 2012_05). Enzyme was set to semi-trypsin, and missed cleavage was 1. No fixed modifications were added. Instead oxidation of methionine and proline were included as variable modifications. The MS tolerance and MS/MS tolerance were 10 ppm and 0.4 Da, respectively. Duo, triple, and quadric charges of precursor ions were selected and an ESI-QUAD-TOF system was set as the instrument used. All data sets were simultaneously searched against the decoy database to apply a false discovery rate (FDR) between 1.5 to 7% and a p-value < 0.01. Peptides with an ion score lower than 30 were rejected. Peptide spectra for protein hits identified by only one peptide were manually inspected, thereby reducing the FDR value. In these cases, only spectra with at least three consecutive y or b ions with high intensities were considered valid.
  • SpeciesList: scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
  • ModificationList: monohydroxylated residue
  • Instrument: instrument model: TripleTof 5600 AB Sciex

Dataset History

VersionDatetimeStatusChangeLog Entry
02013-06-28 13:30:29ID requested
12013-12-13 09:13:30announced
12013-12-16 01:39:02announced
22014-07-28 00:40:10announcedUpdated project metadata.

Publication List

  1. Dataset with its publication pending

Keyword List

  1. submitter keyword: Cornea, laser capture microdissection, lattice corneal dystrophy, paraffin embedded tissue, plaque proteome

Contact List

    Ebbe Toftgaard Poulsen
    • contact affiliation: Department of Molecular Biology and Genetics
    • contact email: etp@mb.au.dk
    • dataset submitter:

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