Identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate–polyacrylamide gel electrophoresis/Western blotting/matrix-assisted laser desorption time-of-flight mass spectrometry

Abstract Background It is important to explain target proteins for the understanding of the pathogenesis of vitreoretinal diseases. In a previous study, we identified more than 100 proteins including seven angiogenic-modulated factors in vitreous humours (VHs). Although there have been many reports...

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Bibliographic Details
Published in:Annals of clinical biochemistry 2008-05, Vol.45 (3), p.307-312
Main Authors: Mukai, Noriko, Nakanishi, Toyofumi, Shimizu, Akira, Takubo, Takayuki, Ikeda, Tsunehiko
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Blotting, Western
Diabetic Retinopathy - metabolism
Electrophoresis, Polyacrylamide Gel
Humans
Molecular Sequence Data
Phosphoproteins - analysis
Phosphoproteins - chemistry
Phosphoproteins - isolation & purification
Phosphotyrosine - analysis
Proteomics - methods
Retinal Detachment - metabolism
Retinal Diseases - metabolism
Retinal Perforations - metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Vitreoretinopathy, Proliferative - metabolism
Vitreous Body - chemistry
Vitreous Body - metabolism
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Summary:Abstract Background It is important to explain target proteins for the understanding of the pathogenesis of vitreoretinal diseases. In a previous study, we identified more than 100 proteins including seven angiogenic-modulated factors in vitreous humours (VHs). Although there have been many reports of expressed protein profiles in VHs, only a few of these are modified proteins, such as those undergoing phosphorylation and oxidation. Methods We applied Western blotting (WB), selective staining of phosphoproteins and mass spectrometry to detect and identify phosphoproteins in VHs of patients with vitreoretinal diseases. After the removal of albumin and immunoglobulins A/G in VHs, the proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and reacted with anti-PY-20 monoclonal antibody on transfer membranes, and treated proteins were visualized with Phos-tag™ and identified by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS). Results WB analysis detected four positive bands, 20, 30, 35 and 55 kDa, in VHs of patients with vitreoretinal diseases. One of them, 55 kDa, was frequently detected in VHs of patients with macular hole (MH) and retinal detachment (RD), but the band was not found in patients with proliferative diabetic retinopathy (PDR). α-1 antitrypsin (α-1 AT) was identified in excised gel pieces of this band. Conclusions We identified five phosphorylated proteins such as α-1 AT in VHs of patients with vitreoretinal diseases by MALDI-TOFMS and WB analysis. Phosphotyrosyl α-1 AT was neither detected in PDR patients nor in any plasma. Phosphotyrosyl α-1 AT may be a new biomarker of MH and RD.
ISSN:0004-5632
1758-1001
DOI:10.1258/acb.2007.007151