Annals of Clinical Biochemistry > Volume 45, Number 3 > Pp. 307-312

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Original Articles

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

Noriko Mukai¹, Toyofumi Nakanishi², Akira Shimizu², Takayuki Takubo² and Tsunehiko Ikeda¹

¹ Department of Ophthalmology; ² Clinical and Laboratory Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki-city, Osaka 569-8686, Japan

Corresponding author: Dr Toyofumi Nakanishi. Email: nakanisi{at}poh.osaka-med.ac.jp

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^TM 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). -1 antitrypsin (-1 AT) was identified in excised gel pieces of this band.

Conclusions: We identified five phosphorylated proteins such as -1 AT in VHs of patients with vitreoretinal diseases by MALDI-TOFMS and WB analysis. Phosphotyrosyl -1 AT was neither detected in PDR patients nor in any plasma. Phosphotyrosyl -1 AT may be a new biomarker of MH and RD.

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