Electrospray Ionisation Mass Spectrometry of Lens Crystallins: Verification and Detection of Errors in Protein Sequences of Bovine γ-Crystallins

Individual bovine gamma crystallins have been isolated by gel permeation chromatography (GPC) followed by fast protein ion-exchange liquid chromatography (FPLC). Electrospray ionisation (ESI) mass spectrometric examination of the isolated proteins confirms the relative molecular masses (RMM) of three γ-crystallins, i.e. γS, γII (or γB) and γIIIb. The RMM of γIVa and γIIIa, however, are inconsistent with the reported sequences of these proteins. Reduction and carboxymethylation followed by ESI mass spectrometric analysis is shown to be a rapid and convenient method for determining the sulphydryl content of these proteins. In the case of γIIIa and γIVa, this showed these proteins each contain five cysteine residues, compared with ten and six, respectively, in the published sequences. The number of cysteine residues is significant since intramolecular disulphide bonds may have a marked effect on the tertiary structures of these proteins. In addition, the RMM of a protein that we have tentatively assigned as γIVb, which has not been sequenced, is reported here. This study demonstrates the considerable advantages offered by ESI mass spectrometry for confirming published amino acid sequences and detecting sequence errors.