Typical 2D-DIGE workflow for comparative expression proteomics. The samples are labelled by fluorescent dyes (Cy 2, 3 and 5) and combined together prior to the IEF. The scanned fluorescent images are analysed by DeCyder™ analysis software using the normalisation by Cy2-labelled pool sample. The protein spots (e.g., crystallin mu) with statistical significance that express different expression profiles between the two groups are further analysed by MALDI-TOF-MS. The peptide mass fingerprint of crystallin (mu) shows the peptides marked with '*' matched to MASCOT search against the NCBInr primate database. The x- and y-axes show the mass to changed (m/z) ratio and the % abundance of the tryptic peptide fragments, respectively. Also depicted in the inset for crystallin (mu) mass spectrum is the representative 3-D protein-expression profiles for the two groups. Typically, the most-abuntant peptide fragment is sequenced by, additive series of the y- and b-ions generated as well as the immonium ions, MALDI-TOF-TOF (e.g., peptide fragment with m/z of 1,667.8) to confirm the identification of protein and charactirise the sites of PTM. Reprinted from Tannu and Hemby  with permission of Elsevier.