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 heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(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 nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight 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 ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.
Fridriksson
E K
EK
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Beavil
A
A
Holowka
D
D
Gould
H J
HJ
Baird
B
B
McLafferty
F W
FW
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007139:Immunoglobulin epsilon-Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010449:Peptide Mapping; D015202:Protein Engineering
D000596:Amino Acids; D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D007139:Immunoglobulin epsilon-Chains; D007073:Immunoglobulin E