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IEF-CE Coupled to MALDI-TOF-TOF MS - A Study of Biomarkers in Aqueous Humor from Persons with Pseudoexfoliations (PEX)

Emilia Hardenborg¹, Aida Zuberovic¹, Amelie Botling Taube², Marit Andersson¹, Albert Alm², Jonas Bergquist¹

Correspondence address: Emilia Hardenborg, Institute of Physical and Analytical Chemistry, P.O.Box 599, Uppsala, 751 24 Sweden.

Keywords: Electrophoresis Capillary; MALDI; MS/MS; Proteomic.

Novel aspect: A new approach of analysing of proteins in aqueous humor using CIEF-CE-MALDI-TOF/TOF MS.

 

Primary open angle glaucoma (POAG) is an age-related disease, which can lead to blindness, affecting about 1 % of people over 40. Pseudexfoliations (PEX), white fibrillar material deposited within the eye, increase the risk of developing POAG. The protein content of aqueous humor can give important information of proteins involved in the formation of PEX in the eye. Kuchle et al.¹ used gel electrophoresis for analysis of total protein content in eyes with and without PEX. They showed that the total protein content in eyes with PEX was twice as high as in eyes without PEX 0.4 mg/mL and 0.2 mg/mL respectively. Berlau et al.² on the other hand did not see this difference in protein content between PEX eyes and non-PEX eyes.

In this study modern techniques for separation and analysis of the protein content of aqueous humor have been used. Isoelectric focusing (IEF) in capillary format in combination with capillary electrophoresis coupled off line to Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (CE-MALDI-TOF-TOF-MS) was used for the analysis of aqueous humor samples collected from patients during cataract surgery. The small sample volumes needed for analysis with capillary electrophoresis is valuable since the sample volumes are limited. By coating the capillaries with a polyamine (PolyE-323), adsorption of proteins to the capillary wall could be minimized. Mass spectrometry adds valuable information of the sample by giving molecular weight of the analytes and by using MS/MS-data structural information can be obtained. The approach of analyzing body fluids with separation by CE and detection with MALDI-TOF-TOF-MS was successful in the analysis and detection of differences in protein content between the two groups of samples analyzed. Aqueous humor from patients with and without PEX was analyzed. Identification of proteins and differences between the two groups will be shown.

References:
1. M. Kuchle, T. S. Ho, N. X. Nguyen, E. Hannappel and G. O. H. Naumann, Invest. Ophthalmol. Vis. Sci. 35, 748 (1994).
2. J. Berlau, P. Lorenz, R. Beck, J. Makovitzky, U. Schlotzer-Schrehardt, H. J. Thiesen and R. Guthoff, Graef. Arch. Clin. Exp. Ophthalmol. 239, 743 (2001).