As seen before from 2-DE analysis, 25 proteins of a total of 90 different proteins identified were found in multiple spots on several positions on the gel. For example, two spots were found at the same M
r but separated by approximately 0.5 pH units (N62,N63 cathepsin D; N31,N35 F1-ATPase-β chain), and one protein was found at different M
rs but with the same pI (C4,C18; glucosidase II). Finally, four proteins were identified at slightly different M
rs and pIs (N58,N61 CRALBP; N5,N7 mitofilin; C45,C46 14-3-3 protein; C41,C42,C43 tropomyosin alpha, tropomyosin 4, tropomyosin alpha 3 chain.), suggesting the presence of different isoforms. These patterns clearly differed from a more frequently observed configuration, where spots were aligned like strings of pearls. This was observed for 15 proteins, which are represented with a series of two to three different spots, close together in a row (e.g., N14,N15,N19 annexin VI; N27,N28 S-arrestin; N22,N23 RPE65; C29,C30 α-enolase; C32,C33 CK 18). Such a series of spots is probably due to glutamine deamidation, chemically induced during the 2-DE procedure and resulting in differentially charged proteins rather than different protein isoforms.
39 We also mapped one protein (N1 cathepsin D) at a position on the gel that indicated a greater M
r than predicted and another two spots (C11 LIM-motif containing protein kinase; C31 eIF-4A
I) appeared at a pI different from the predicted one. In addition, retinal pigment epithelial membrane receptor (N34) was annotated at a position that differed in M
r and pI from the predicted values. Computer assisted sequence analysis identified this protein as a bovine isoform of RPE65, in this case, probably representing a partially processed stable fragment with different M
r and pI.