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News
No. B113
MALDI-TOF Mass Spectrometry
Glycosylation Profile of IgGs Using a Linear Benchtop
MALDI-TOFMS and Affinity Purification of Fc
Glycosylation on protein plays wide-range vital roles in
biological processes from the stabilization of protein
conformation to the expression of binding specificity.
In this view, a characterization of the N-/O-linked
glycan is rather significant, especially in the
development of biopharmaceuticals. To date, whereas
intensive efforts have been conducted on the precise
characterization of glycans with high-end mass
spectrometers, inexpensive easy-to-use commercial
instruments have been anticipated for batch analysis
assuming screening or quality assurance/quality
control (QA/QC). A newly developed bench-top
MALDI-TOFMS, MALDI-8020, is expected to be a
suitable instrument in terms of sufficient specification,
through-put, and cost effectiveness. We attempted to
characterize the glycosylation of IgG using the MALDI-
8020 without a release of glycan. To this end, we
examined a quick preparation using affinity beads and
enzymatic cleavage.
Y.Yamazaki
Preparation of Fc
A work-flow of Fc affinity separation is shown in Fig. 1.
NIST mAb, IgG from mouse serum, and myeloma IgG
were subject to MS analysis. IdeZ and Protein-A
magnetic beads (Mag Sepharose™) were purchased
from Promega and GE Healthcare, respectively. The
IgGs dissolved in tris-NaCl buffer were incubated with
IdeZ at 37 °C for two hours. Resultant solutions
including Fc and F(ab’)2 were applied to the magnetic
beads equilibrated with the tris-buffer, then washed
with the same buffer several times. The Fc associated
with Protein-A was eluted with acidic solution, then
desalted with ZipTip® C18.
MALDI-TOFMS
All MS analysis in positive ion mode was performed
with a bench-top linear MALDI-TOFMS (MALDI-8020).
Sinapinic acid (SA) and Ferulic acid (FA) were applied to
MS analysis as matrix at the same condition as follows;
20 mg/mL in 50 % acetonitrile/milliQ water with 0.1 %
TFA.
Statistical Data Analysis
eMSTAT Solution™ (Shimadzu Corp., Japan) was applied
to perform classification/differentiation of glycosylated-
Fc regions.
Fig. 1 Work-Flow of Fc Affinity Separation/Detection
Results
Fig. 2 illustrates the mass spectra of whole IgGs. At
nearly 150 kDa, their molecular weights could be easily
observed. However, almost no information relating to
glycosylation was obtained due to insufficient mass
resolving power in this high m/z range .
Subsequently, all IgGs were subjected to the work-flow
shown in Fig. 1 to purify Fc regions that retain varieties
of glycosylation. Molecular weights of the purified Fc
were nearly 25 kDa, in which the MS resolving power of
MALDI-8020 is sufficient to recognize the varieties of
glycosylation.
