Mass spectroscopic identification of in vitro glycated sites of MIP.
OBJECTIVE
Earlier reconstitution studies showed that glycation of the major intrinsic peptide (MIP) of the lens affected the permeability of liposomes. This study is aimed to identify in vitro glycated sites.


METHODS
Urea- and alkali-washed calf lens membranes were incubated with 0 and 1 M glucose for 5 days. Following the incubation, MIP was purified by size exclusion HPLC. The C-terminus peptide of MIP was then cleaved by cyanogen bromide (CNBr) and purified by C4 reversed-phase HPLC. The CNBr peptide was analyzed, either directly or after digestion with trypsin, by electrospray ionization mass spectrometry (ESIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).


RESULTS
The ESIMS and MALDI-MS analyses of the intact C-terminus peptide from 1 M glucose incubated samples showed a mass shift equivalent to one, two and three glucose adducts. The MALDI-MS of the tryptic digest of the same sample showed peptides with mass shift equivalent to one or more glucose adducts. Sequence assignment confirmed that these glycated peptides contained lys238 and lys259. Although the intact C-terminus peptide showed up to three glucose adducts, we could not assign any tryptic peptide that contained glycated lys228. Samples incubated with 0 M glucose did not show any protein modifications.


CONCLUSIONS
The data suggest that in vitro glycation sites of calf lens MIP are lys238 and lys259, and possibly lys228.
Swamy-Mruthinti
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Department of Biochemistry & Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Schey
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KL
D000818:Animals; D020346:Aquaporins; D001665:Binding Sites; D002417:Cattle; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007908:Lens, Crystalline / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008566:Membranes / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0