Automated Glycan Sequencing from Tandem Mass Spectra of N‑Linked Glycopeptides

Mass spectrometry
has become a routine experimental tool for proteomic
biomarker analysis of human blood samples, partly due to the large
availability of informatics tools. As one of the most common protein
post-translational modifications (PTMs) in mammals, protein glycosylation
has been observed to alter in multiple human diseases and thus may
potentially be candidate markers of disease progression. While mass
spectrometry instrumentation has seen advancements in capabilities,
discovering glycosylation-related markers using existing software
is currently not straightforward. Complete characterization of protein
glycosylation requires the identification of intact glycopeptides
in samples, including identification of the modification site as well
as the structure of the attached glycans. In this paper, we present
GlycoSeq, an open-source software tool that implements a heuristic
iterated glycan sequencing algorithm coupled with prior knowledge
for automated elucidation of the glycan structure within a glycopeptide
from its collision-induced dissociation tandem mass spectrum. GlycoSeq
employs rules of glycosidic linkage as defined by glycan synthetic
pathways to eliminate improbable glycan structures and build reasonable
glycan trees. We tested the tool on two sets of tandem mass spectra
of N-linked glycopeptides cell lines acquired from breast cancer patients.
After employing enzymatic specificity within the N-linked glycan synthetic
pathway, the sequencing results of GlycoSeq were highly consistent
with the manually curated glycan structures. Hence, GlycoSeq is ready
to be used for the characterization of glycan structures in glycopeptides
from MS/MS analysis. GlycoSeq is released as open source software
at https://github.com/chpaul/GlycoSeq/.