Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS

High-throughput mass
spectrometry (MS) glycomics is an emerging
field driven by technological advancements including sample preparation
and data processing. Previously, we reported an automated protocol
for the analysis of N-glycans released from plasma
proteins that included sialic acid derivatization with linkage-specificity,
namely, ethylation of α2,6-linked sialic acid residues and lactone
formation of α2,3-linked sialic acids. In the current study,
each step in this protocol was further optimized. Method improvements
included minimizing the extent of side-reaction during derivatization,
an adjusted glycan purification strategy and mass analysis of the
released N-glycans by ultrahigh resolution matrix-assisted
laser desorption/ionization Fourier transform ion cyclotron resonance
MS. The latter resolved peak overlap and simplified spectral alignment
due to high mass measurement precision. Moreover, this resulted in
more confident glycan assignments and improved signal-to-noise for
low-abundant species. The performance of the protocol renders high-throughput
applications feasible in the exciting field of clinical glycomics.