MoP-16


LC-MS AND MS/MS ANALYSIS OF AGE-RELATED MODIFICATIONS TO HUMAN CRYSTALLINS

Glen M. Stutchbury, Allan Weimann, Margaret M. Sheil and Roger J. W. Truscott

The Australian Catract Research Foundation
Department of Chemistry, The University of Wollongong, Wollongong 2522


Electrospray ionisation mass spectrometry (ESI-MS) and liquid chromatography (LC-MS) has been employed extensively for the analysis of intact lens crystalline, the major structural proteins of the eye [1]. This work is largely focussed on crystallins from normal lenses and their tryptic fragments. By contrast, very little work has been carried out on the corresponding crystallins after the modifications involved in cataract formation. Analysis of intact crystallins obtained from cataractous lens tissues is very difficult as the fifteen crystallins have the added complexity of a variety of potential post-translational modifications. These include: the oxidation of cysteine to either intramolecular disulphides or intermolecular disulphides resulting in aggregation of the lens proteins; oxidation of methionine to methionine sulphoxide; addition of a chromophore; proteolytic cleavage; phosphorylation and glycation [2]. As there is no protein turnover in the lens, lens crystallins are as old as the individual and during this period it is conceivable that multiple modifications might occur. The arrangement of the crystallins within the lens affords the high refractive index required for visual acuity. Hence, maintenance of crystallin solubility is crucial for the transmission of light. The development of disulphides has the potential to affect both of these characteristics of the lens and it has been observed that up to 90% of all cysteine residues are oxidised in senile nuclear cataract.

This paper describes the development of a differential alkylation technique to facilitate the identification of the thiol oxidation state of the crystallins. These modified proteins are enzymatically digested prior to initial screening by LC-MS with an electrospray ionisation mass spectrometer. Tandem mass spectrometry (MS/MS) has been used to sequence modified peptides allowing identification of peptides as well as the sites of cysteine modification.

  1. Kilby G. W., Truscott R. J. W., Stutchbury G. M. and Sheil M. M. Rapid Commun. Mass Spectrom. 1996. 10, 123-129.
  2. Harding J. J. Cataract: Biochemistry, Epidemiology and Pharmacology. Chapman and Hall London.