Proteomic analysis of exfoliation deposits.
OBJECTIVE
To increase knowledge of the biochemical composition of lenticular exfoliation material (XFM) by using proteomic approaches.


METHODS
Anterior lens capsules from patients with and without exfoliation syndrome (XFS) were homogenized in formic acid and subjected to cyanogen bromide (CNBr) cleavage, and the pattern of chemically generated fragments was compared by SDS-PAGE after silver staining. Unique XFS bands not present in control cases were excised, digested with TPCK-trypsin, and the resultant peptides sequenced with quadrupole time-of-flight mass spectrometry (MS). In parallel experiments, CNBr-fragmented XFM was separately digested in solution with trypsin and elastase, and the resultant peptide mixture was analyzed by liquid chromatography coupled to tandem MS followed by identification through homology searches at nonredundant protein databases. Immunolocalization of the MS-identified components were performed in XFS versus control samples by using conventional immunohistochemical methods and light microscopy.


RESULTS
In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid P-component, which are well-known extracellular matrix and basement membrane components of XFM, the proteomic approaches identified the multifunctional protein clusterin and tissue inhibitor of metalloprotease (TIMP)-3 as well as novel molecules, among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q. In all cases, classic immunohistochemistry confirmed their location in XFM.


CONCLUSIONS
A novel solubilization strategy combined with sensitive proteomic analysis emphasizes the complexity of the XFS deposits and opens new avenues to study the molecular mechanisms involved in the pathogenesis and progression of XFS.
Ovodenko
Boris
B
Department of Ophthalmology, New York Eye and Ear Infirmary, New York, NY, USA.
Rostagno
Agueda
A
Neubert
Thomas A
TA
Shetty
Vivekananda
V
Thomas
Stefani
S
Yang
Austin
A
Liebmann
Jeffrey
J
Ghiso
Jorge
J
Ritch
Robert
R
D000368:Aged; D000369:Aged, 80 and over; D002387:Cataract Extraction; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D017889:Exfoliation Syndrome / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007903:Lens Capsule, Crystalline / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008875:Middle Aged; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D020543:Proteome