In-gel digestion and LC-MS/MS identification of protein bands detected on the gel
As shown in Figure 6, 6 bands were excised and a blank between band B1 and B2 were also excised as background band (BB). All seven bands were in-gel digested and proteins in those band were identified by LC-MS/MS as described in Materials and Methods. A total of 526 proteins were identified in all bands excised with 277 proteins were identified in B1 bands, 272 proteins were identified in B2 bands, 330 in B3 bands, 380 in B4 bands, 264 in B5 bands, and 301 proteins in B6 bands, and 325 proteins in BB. The mass spectrometry identification covers 5 magnitude of order in dynamic range, with a few abundant proteins accounting for a higher % of total protein identified and more proteins are at much lower abundance. The distribution of proteins identified in each band along with their accumulative abundance were plotted in Figure 7. Proteins accounting for more than 1% of total proteins identified in that band based on the total peptide ion intensity are ranked and their accumulative abundance plotted in Figure 7 (based on K12 database). Based on this ranking and a research of BL21 database and the band intensity quantitation based on Coomassie blue stained gel, the top abundant proteins that have estimated total amount over 10 ppm (ng/mg) were listed in Table 3, along with their pI, MW and in silico immunogenicity score and subjective risk.