ac202995x_si_001.pdf (63.58 kB)
Rapid Identification of Low Level Glycation Sites in Recombinant Antibodies by Isotopic Labeling with 13C6-Reducing Sugars
journal contribution
posted on 2012-03-06, 00:00 authored by Jennifer Zhang, Taylor Zhang, Lihua Jiang, Daniel Hewitt, YungFu Huang, Yung-Hsiang Kao, Viswanatham KattaRecombinant antibodies exhibit low levels of glycation
from exposure
to reducing sugars during production. As the glycation sites are typically
distributed across the entire antibody, the levels at any one site
are low and it becomes difficult to detect them in the conventional
peptide maps. A model antibody was subjected to forced glycation by
incubating with a high concentration of a 1:1 mixture of 12C6/13C6 reducing sugars with the
assumption that the same sites in the native antibody will be glycated
but to a lower extent. This approach simplified the detection of glycated
tryptic peptide elution in the LC/MS analysis by giving a unique signature
of two molecular ions with equal intensity and differing by 6.018
Da. An in-house developed script automatically processed large data
files to generate a list of such peptide mass pairs. The high mass
accuracy of the Orbitrap allowed us to assign the sequences unambiguously
by comparison with all possible glycated peptide masses. This sequence
list was subsequently used to verify their presence/absence in the
digest of the native antibody. This work flow enabled rapid and confident
identification of site-specific glycation even when levels are below
0.5%. We found the glycation sites to be distributed across the entire
antibody studied.