In-Depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC–MS/MS

This
work presents a detailed analysis of glycopeptides produced
in the tryptic digestion of an IgG1 reference material. Analysis was
done by nanospray ESI LC–MS/MS over a wide range of HCD collision
energies with both conventional 1D separation for various digestion
conditions and a 20 fraction 2D-LC study of a single digest. An extended
version of NIST-developed software for analysis of “shotgun”
proteomics served to identify the glycopeptides from their precursor
masses and product ions for peptides with up to three missed cleavages.
A peptide with a single missed cleavage, TKPREEQYNSTYR, was dominant
and led to the determination of almost all glycans reported in this
study. The 2D studies found a total of 247 glycopeptide ions and 60
glycans of different masses, including 30 glycans found in the 1D
studies. This significantly larger number of glycans than found in
any other glycoanalysis of therapeutic glycoproteins is due to both
the improved separation of sialylated versus asialylated species in
the first (high-pH) dimension and the ability to inject large amounts
of glycosylated peptides in the 2D studies. Systematic variations
in retention with glycan size were also noted. Energy-dependent changes
in HCD fragmentation confirmed the proposed glycan structures and
led to a peak-annotated mass spectral library to aid the analysis
of glycopeptides derived from IgG1 drugs.