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.