Masses of guinea pig lens cortical crystallins were determined by electrospray ionization mass spectrometry (ESIMS) after alkylation and elution of the proteins from 2-DE gels. Of a total of 29 protein spots, 15 were found to contain a sufficient amount of protein for ESIMS analysis. Ten of the 15 masses matched with calculated masses based on cDNA sequences, but 5 masses (22433.9 Da, 28065.4 Da, 22610.8 Da, 23802.9 Da, and 22388.7 Da) could not be matched. Six crystallins, which were known to be present in the guinea pig lens based on the cDNA sequences (Table 3), could not be matched with measured masses determined by ESIMS (labeled as ND, not determined). All measured masses were within an instrument error of 0.01%. Theoretical masses were calculated after removing the NH₂-terminal methionine from all sequences (except those for alphaA, alphaAinsert, alphaB and betaA3). A mass unit of 42 was added to the masses of all crystallins (except those for gammaA, gammaB and gammaN) to account for the N-terminal acetyl group. Alkylated protein mass was accounted for by adding 57.1 mass units to each cysteine residue. The asterisk indicates that 16 mu was subtracted based on the assumption of the addition of one oxygen atom.