2 research outputs found
Spatially-Resolved Exploration of the Mouse Brain Glycome by Tissue Glyco-Capture (TGC) and Nano-LC/MS
Tissue glyco-capture (TGC), a highly
sensitive MS-compatible method
for extraction of glycans from tissue, was combined with structure-specific
nano-LC/MS for sensitive and detailed profiling of the mouse brain
glycome. Hundreds of glycan structures were directly detected by accurate
mass MS and structurally elucidated by MS/MS, revealing the presence
of novel glycan motifs such as antennary fucosylation, sulfation,
and glucuronidation that are potentially associated with cellular
signaling and adhesion. Microgram-level sensitivity enabled glycomic
analysis of specific regions of the brain, as demonstrated on not
only brain sections (with a one-dimensional spatial resolution of
20 μm) but also isolated brain structures (e.g., the hippocampus).
Reproducibility was extraordinarily high (<i>R</i> >
0.98)
for both method and instrumental replicates. The pairing of TGC with
structure-specific nano-LC/MS was found to be an exceptionally powerful
platform for qualitative and quantitative exploration of the brain
glycome
Characterization of Site-Specific <i>N</i>‑Glycopeptide Isoforms of α‑1-Acid Glycoprotein from an Interlaboratory Study Using LC–MS/MS
Glycoprotein conformations are complex and heterogeneous. Currently,
site-specific characterization of glycopeptides is a challenge. We
sought to establish an efficient method of <i>N</i>-glycoprotein
characterization using mass spectrometry (MS). Using alpha-1-acid
glycoprotein (AGP) as a model <i>N</i>-glycoprotein, we
identified its tryptic <i>N</i>-glycopeptides and examined
the data reproducibility in seven laboratories running different LC–MS/MS
platforms. We used three test samples and one blind sample to evaluate
instrument performance with entire sample preparation workflow. 165
site-specific <i>N</i>-glycopeptides representative of all <i>N</i>-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP <i>N</i>-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified <i>N</i>-glycopeptides. The relative quantities of the 10 major <i>N</i>-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific <i>N</i>-glycopeptide isoforms of AGP from control and disease plasma sample