Figure 8. A schematic representing the four spots recovered from the two-dimensional gel profiles of the WT αB-crystallin and individual deamidated mutant proteins. These are numbered as spot number 1 to 4. For mass spectrometric analysis, the individual protein spots were excised from a SDS-PAGE gel. After destaining of the individual spots, the samples were washed for 10 min with 25 mM ammonium bicarbonate before digestion with trypsin (12 ng/μl) for 16 h at 37 °C. Peptide solutions were then extracted using 100 μl of a 50:50 mixture of 5% formic acid and acetonitrile for 30 min. Supernatants were collected and evaporated to dryness. Samples were resuspended in 10 μl of 0.1% formic acid and desalted using C-18 ZipTips. The samples were spotted to the MALDI-TOF 96 well gold-coated target plates after mixing with cyano-4-hydroxycinnamic acid (CHCA) matrix. MALDI-TOF MS was performed, and spectra were collected. The identity of the proteins was determined by using the NCBI database.