title: Tear film proteins deposited on high water content contact lenses identified with two-dimensional gel electrophoresis and mass spectrometry.
abstract: OBJECTIVE
  Tear film proteins adhere to the surface of contact lenses (CLs). While the proteins in the tears have been extensively studied with various proteomic techniques, adhered proteins to CLs are less studied. In this pilot study, we have separated proteins with 2D gel electrophoresis prior to the conventional mass spectrometry (MS) in order to analyse the deposited proteins on hydrogel CLs from myopic patients.
  
  
  METHODS
  pHEMA and PVA hydrogel CLs worn by 3 patients for different time lengths were analysed. After wear, the CLs were frozen at -20°C. Proteins were extracted in lysis buffer, separated on 12% polyacrylamide gels and silver-stained. Protein spots were excised and identified with liquid chromatography - tandem MS.
  
  
  RESULTS
  Deposited proteins were extracted with a yield of 26-66 μg and separated by 2D gel electrophoresis. The silver-stained gels showed similar protein patterns independent of the patient, hydrogel type and wear time. Seventy-two spots were analysed with MS, representing at least 12 different tear film proteins or protein fragments.
  
  
  CONCLUSIONS
  Deposited tear film proteins from a single set of CLs worn for 1 day can successfully be analysed first with 2D gel electrophoresis and subsequently with MS, thus making examination of individual patients possible. The protein composition appeared homogeneous between the test persons which is a necessity for additional comparison analysis. The molecular masses of the identified proteins indicate that protein degradation occurs only as a minor event. Myopic patients were investigated in this pilot study, but the combined techniques can easily be applied to other eye diseases.
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Aarhus C, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Nielsen
authors.affiliation: Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Vorum
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Aarhus C, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Ehlers
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Aarhus C, Denmark.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Aagaard
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Aarhus C, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Hjortdal
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
authors.forename: Bent
authors.initials: B
authors.lastname: Honoré
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid; D003263:Contact Lenses, Hydrophilic; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D009216:Myopia / Q000628:therapy; D010865:Pilot Projects; D011485:Protein Binding; D053719:Tandem Mass Spectrometry; D013666:Tears / Q000737:chemistry
chemical_list: D005136:Eye Proteins; C042434:tear proteins
keywords: 2D gel electrophoresis; contact lenses; deposited proteins; mass spectrometry; tear film