Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural he terogeneity, multiple glycosylation sites, and different forms. Here, a top -down mass spectrometry (MS) characterization is applied to three construct s of the Fc segment of IEE: Fc epsilon(3-4) (52 kDa) and Fc epsilon(2-3-4)( 2) (76 kDa) disulfide-bonded homodimers, Fourier transform MS of a reduced sample of Fc epsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851 , and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduc ed Fc epsilon(2-3-4)2 protein yielded a peptide mixture with molecular weig ht values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular io n values. The 2-D mass spectrum of two glycosylated peptides showed these t o have high-mannose structures, -GlcNAc-(hex)(n), demonstrating that Fc eps ilon(2-3-4) has a single such structure of n = 5-9, For a mutated sample of Fc epsilon(3-4), in addition to five glycoforms, MS showed a molecular dis crepancy that could be assigned with proteolysis and 2-D mass spectra to th e oxidation of two methionines and an additional residue difference.