Isolation and structural determination of human lens alpha-crystallins by chromatography and mass spectrometry

Cataract, an opacity of the lens, is the major cause of blindness worldwide. Lens α-crystallins are not only structural proteins important to the maintenance of lens transparency, but also molecular chaperones that suppress the aggregation of other proteins. Post-translational modification of α-crystallins may result in opacification of the eye lens by disrupting the order of crystallin packing and/or by interfering with the chaperone activity. To obtain α-crystallins, lenses were homogenized in a buffer and centrifuged. The supernatant was fractionated by size exclusion chromatography to isolate the α-crystallins from the other lens crystallins. The α-crystallins were fractionated into αA and αB by reversed phase HPLC. Each of these groups of proteins was further isolated by anion exchange chromatography. The proteins\u27 retention time in anion exchange chromatography and their molecular weights, determined by ESIMS indicated the presence of deamidation, phosphorylation, C-terminal degradation, acetylation, and some unknown modifications. Multiple modifications occurred on the same molecule. Each isolated protein was enzymatically digested. The peptides in the digests were fractionated and analyzed by on-line microbore and/or capillary reversed phase HPLC-ESIMS. Unknown modified peptide was further analyzed by MS/MS to determine the exact location of the modified residue. To identify the elemental composition of modification, the exact mass of modified peptide was determined using a resolution of 10,000. Both MS/MS and exact mass analyses were performed using a FAB source. Identified modifications not previously reported included an additional site of phosphorylation—Ser 53 of αB, acetylation. of Lys 70 of αA, and an unknown addition of 72 Da. to the αB. For this unknown modification, determination of the exact mass provided the elemental composition of C 3H5O2. Data from MS/MS analysis indicated that the modification was at the C-terminal Lys of αB. Acetylation of the modified peptide suggested that the 72 Da adduct was at the α-amino group of C-terminal Lys of αB. It is possible that the modification is the product of an enzymatic-mediated reaction of lactic acid or transglutaminase mediated reaction of lactoyl amide