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T. Moller-Pedersen, C.J. Hedegaard, R.B. Andersen, I.B. Thogersen, G.K. Klintworth, J.J. Enghild; TGFBIp (BIGH3) in Granular Corneal Dystrophy Type III (Reis-Bücklers) - Identification Using Mass Spectrometry of 15-Year-Old Two-Dimensional Protein Gels . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3863.
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Purpose: To investigate the accumulation and composition of transforming growth factor-ß induced protein (TGFBIp) in granular corneal dystrophy type III (GCDIII; Reis-Bücklers) and to demonstrate the ability to perform mass spectrometry on old two-dimensional protein gels. Methods: The cornea of a patient with GCDIII was obtained during penetrating keratoplasty. A molecular genetic analysis of the patient and several affected family members revealed a R124L mutation in exon 4 of the TGFBI (BIGH3) gene. In 1987, 2D-gel electrophoresis was performed on proteins extracted from the GCDIII affected cornea, while an age-matched normal human cornea served as control. The gels were kept at room temperature until year 2002 where protein spots of interest were cut out and digested with trypsin. The tryptic derived peptides were analyzed using mass spectrometry. Results: Four different proteins (TGFBIp, aldehyde dehydrogenase class 3, actin, and albumin) were identified in the 15-year-old gels. Using image analysis, the amount of TGFBIp was found to be 30 to 50-fold higher in the GCDIII affected cornea relative to the normal tissue. In both situations, TGFBIp migrated on the 2D-gels as a 63-kDa protein. Mass spectrometry revealed the same nine peptides in both the normal and the GCDIII affected cornea, including one peptide situated at the NH2-terminal. Moreover, the GCDIII affected cornea contained a large amount of 40-kDa TGFBIp fragments that were lacking sequences in both the NH2- and COOH-terminal (including the RGD-sequence). Visit www.corneaproteomics.org for more information. Conclusions: Mass spectrometry can be performed on old two-dimensional protein gels. In both normal and GCDIII affected corneas, the majority of TGFBIp migrates on 2D-gels as a 63-kDa protein with an intact NH2-terminal. However, 40-kDa TGFBIp fragments can also be identified. The amount of TGFBIp is approximately 30 to 50-fold higher in the GCDIII affected cornea relative to the healthy control.
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