Abstract
Protein glycosylation is a common and highly heterogeneous post-translational modification that challenges biophysical characterization technologies. The heterogeneity of glycoproteins makes their structural analysis difficult; in particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) often suffers from poor sequence coverage near the glycosylation site. A pertinent example is the Fc gamma receptor RIIIa (FcγRIIIa, CD16a), a glycoprotein expressed on the surface of natural killer cells (NK) that binds the Fc domain of IgG antibodies as a trigger for antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we describe an adaptation of a previously reported method using PNGase A for post-HDX deglycosylation to characterize the binding between the highly glycosylated CD16a and IgG1. Upon optimization of the method to improve sequence coverage while minimizing back-exchange, we achieved coverage of four of the five glycosylation sites of CD16a. Despite some back-exchange, trends in HDX are consistent with previously reported CD16a/IgG-Fc complex structures; furthermore, binding of peptides covering the glycosylated asparagine-164 can be interrogated when using this protocol, previously not seen using standard HDX-MS.