Mass spectrometry (MS) can unlock crucial insights into
the intricate
world of glycosylation analysis. Despite its immense potential, the
qualitative and quantitative analysis of isobaric glycopeptide structures
remains one of the most daunting hurdles in the field of glycoproteomics.
The ability to distinguish between these complex glycan structures
poses a significant challenge, hindering our ability to accurately
measure and understand the role of glycoproteins in biological systems.
A few recent publications described the use of collision energy (CE)
modulation to improve structural elucidation, especially for qualitative
purposes. Different linkages of glycan units usually demonstrate different
stabilities under CID/HCD fragmentation conditions. Fragmentation
of the glycan moiety produces low molecular weight ions (oxonium ions)
that can serve as a structure-specific signature for specific glycan
moieties; however, the specificity of these fragments has never been
examined closely. Here, we particularly focused on N-glycoproteomics
analysis and investigated fragmentation specificity using synthetic
stable isotope-labeled N-glycopeptide standards. These standards were
isotopically labeled at the reducing terminal GlcNAc, which allowed
us to resolve fragments produced by the oligomannose core moiety and
fragments generated from outer antennary structures. Our research
identified the potential for false-positive structure assignments
due to the occurrence of “Ghost” fragments resulting
from single glyco unit rearrangement or mannose core fragmentation
within the collision cell. To mitigate this issue, we have established
a minimal intensity threshold for these fragments to prevent misidentification
of structure-specific fragments in glycoproteomics analysis. Our findings
provide a crucial step forward in the quest for more accurate and
reliable glycoproteomics measurements.