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

Ann Clin Biochem 2008;45:307-312
doi:10.1258/acb.2007.007151
© 2008 Association for Clinical Biochemistry

 

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Original Articles


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 understandingof the pathogenesis of vitreoretinal diseases. In a previousstudy, we identified more than 100 proteins including sevenangiogenic-modulated factors in vitreous humours (VHs). Althoughthere have been many reports of expressed protein profiles inVHs, only a few of these are modified proteins, such as thoseundergoing 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-tagTM and identified by matrix-assisted laser desorptiontime-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). {alpha}-1 antitrypsin ({alpha}-1 AT) was identified in excised gelpieces of this band.

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


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