Quantitative Antibody Profiling with Detailed Structural
Analyses of N-Glycans:
Sequential Disassembly by ITMSn
Andrew Hanneman, David Ashline, Hailong Zhang, and Vernon Reinhold
The University of New Hampshire Glycomics Center and Glycan Connections LLC
Quantitative HPLC profiling of fluorophore tagged N-glycans is often coupled with online
mass spectrometry (MS, MS2) to determine composition and partial sequence
information and additional details uncovered by exoglycosidase treatments. These
approaches can leave unresolved structural issues requiring further investigation
particularly with samples involving glycoengineered products or nontraditional
expression systems. For improved understanding about glycan structural biology
including branching, linkages, and epitopes HPLC peaks may be separately collected,
infused, and studied by nanospray ion trap sequential mass spectrometry (ITMSn), and
including the incorporation of an additional methylation step. HPLC fractions may also
be probed enzymatically prior to methylation and MSn. Direct infusion provides an
enhanced duty cycle to dig deep into structural details transparent to MS2 and
enzymology alone. Facile rupture of the N-linked chitobiose bond in MS2 removes the
fluorescent tag and core fucosylation (if present), leaving abundant product ions to detail
the remaining structure at higher CID stages (MS3-7). Fragments are confirmed by
spectral matching in a library compiled from synthetic standards and well characterized
natural products. Glycan structural reassembly follows from tracking fragmentation
pathways, matching scars and mass values, and use of spectral documentation as a
substitute for unspecified (bracketed) compositions and biological inference. The
described protocols introduce an opportunity to resolve glycan structural details de novo
from bioengineered products where fluorescence profiles or biological data indicate
undefined or aberrant components.