Multiplexed
sample preparation workflow for N-
and O-glycan profiling. Intra- and interday repeatability
of the optimized method. (A) Proteins are immobilized on a polyvinylidene
fluoride (PVDF) membrane by the addition of a (pure) glycoprotein,
cell lysates, or derived from biological material (e.g., plasma). N-Glycans are released by the addition of PNGase F and eluted
from the PVDF membrane. The O-glycans are released
by adding a release agent and eluted from the PVDF membrane followed
by a labeling procedure (2-AB). Eventually, the samples were analyzed
using C18 nano-LC-MS/MS followed by data analysis. (B) Inter- and
intraday repeatability of the O-glycan workflow.
Average relative intensities for the O-glycans are
displayed for those with a relative abundance above 1% per day. Error
bars represent the standard deviations. Graphics in (A) were created
using https://biorender.com/: H, hexose; N, N-acetylhexosamine; F, fucose; S, N-acetylneuraminic acid. Reproduced with permission from
ref (24). Copyright
2022 de Haan et al.