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Carsten Schmelter, Natarajan Perumal, Sebastian Funke, Norbert Pfeiffer, Franz H Grus; Identification and characterization of human tear film glycoproteins. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2489.
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© ARVO (1962-2015); The Authors (2016-present)
The glycosylation patterns of tear film proteins have a major influence on their structure and stability resulting in changes to various protein properties like signaling or regulatory functions. Up to date, tear proteomic glycosylation studies are rare and limited on single or function-related proteins. Therefore, the main objective of this study was to give a comprehensive overview about the glycosylation pattern of the human tear proteome by use of a mass spectrometric platform.
Tears were pooled from 8 healthy volunteers (mean age: 28 ± 3). Individual tear samples were collected by capillary technique and Schirmer’s strips. Two-dimensional gel electrophoresis (2-DE) was performed and the gels were stained with two different dyes: Glycoprotein staining kit to detect glycoproteins and Colloidal Blue Staining to visualize the total protein profile. The tear proteome profiles were analyzed by proteomic based liquid chromatography - mass spectrometry (LC-MS) strategies. By the usage of bioinformatic tools based on de novo sequencing, glycoproteins could be revealed and glycosylation sites characterized.
By 2-DE analysis multiple glycosylation sites of human tear proteins were supported. Due to buttom-up LC-MS analysis we could identify up to 30 proteins (false discovery rate < 1%) displaying a complex glycosylation profile. Thereby, it was possible to detect these glycoproteins in tear samples collected independently by two different approaches. By state-of-the-art bioinformatic analyses, several modification sites could be identified. Thus, for instance, it was possible to detect new glycosylation sites of abundant tear proteins like Ig alpha-1 or Ig alpha-2 chains (constant C regions) as well as modification sites of lesser represented proteins like Cadherin-6 or Ras-specific guanine nucleotide-releasing factor 2.
Our results further demonstrate that the human tear film consists of many proteins which show a complex glycosylation pattern. Aberrant glycosylations of proteins are known to be involved in various pathophysiological processes, providing the idea that glycosylation patterns may be of particular interest in biomarker research. Further studies will be needed to explore the complex modification pattern of the human tear proteome and to evaluate their possible role in the formation of eye disorders.
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