MASS SPECTROSCOPIC IDENTIFICATION OF IN-VITRO GLYCATED SITES OF MIP

Purpose. Earlier reconstitution studies showed that glycation of the m ajor intrinsic peptide (MIP) of the lens affected the permeability of liposomes. This study is aimed to identify in vitro glycated sites. Me thods. Urea- and alkali-washed calf lens membranes were incubated with 0 and 1 M glucose for 5 days. Following the incubation, MIP was purif ied by size exclusion HPLC. The C-terminus peptide of MIP was then cle aved by cyanogen bromide (CNBr) and purified by C4 reversed-phase HPLC . The CNBr peptide was analyzed, either directly or after digestion wi th trypsin, by electrospray ionization mass spectrometry (ESIMS) and m atrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS ). Results. The ESIMS and MALDI-MS analyses of the intact C-terminus p eptide from 1 M glucose incubated samples showed a mass shift equivale nt to one, two and three glucose adducts. The MALDI-MS of the tryptic digest of the same sample showed peptides with mass shift equivalent t o one or more glucose adducts. Sequence assignment confirmed that thes e glycated peptides contained lys238 and lys259. Although the intact C -terminus peptide showed up to three glucose adducts, we could not ass ign any tryptic peptide that contained glycated lys228. Samples incuba ted with 0 M glucose did not show ally protein modifications. Conclusi ons. The data suggest that in vitro glycation sites of calf lens MIP a re lys238 and lys259, and possibly lys228.