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Linkage-Specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of IgG Glycopeptides
journal contribution
posted on 2015-08-18, 00:00 authored by Noortje de Haan, Karli
R. Reiding, Markus Haberger, Dietmar Reusch, David Falck, Manfred WuhrerGlycosylation
is a common co- and post-translational protein modification,
having a large influence on protein properties like conformation and
solubility. Furthermore, glycosylation is an important determinant
of efficacy and clearance of biopharmaceuticals such as immunoglobulin
G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight
(TOF)-mass spectrometry (MS) shows potential for the site-specific
glycosylation analysis of IgG at the glycopeptide level. With this
approach, however, important information about glycopeptide sialylation
is not duly covered because of in-source and metastable decay of the
sialylated species. Here, we present a highly repeatable sialic acid
derivatization method to allow subclass-specific MALDI-TOF-MS analysis
of tryptic IgG glycopeptides. The method, employing dimethylamidation
with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide
(EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different
masses for the functionally divergent α2,3- and α2,6-linked
sialic acids. Respective lactonization and dimethylamidation leads
to their direct discrimination in MS and importantly, both glycan
and peptide moieties reacted in a controlled manner. In addition,
stabilization allowed the acquisition of fragmentation spectra informative
with respect to glycosylation and peptide sequence. This was in contrast
to fragmentation spectra of underivatized samples, which were dominated
by sialic acid loss. The method allowed the facile discrimination
and relative quantitation of IgG Fc sialylation in therapeutic IgG
samples. The method has considerable potential for future site- and
sialic acid linkage-specific glycosylation profiling of therapeutic
antibodies, as well as for subclass-specific biomarker discovery in
clinical IgG samples derived from plasma.