A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C‑ and O‑Mannosylated Peptides

Mass
spectrometry (MS) easily detects C-mannosylated peptides from
purified proteins but not from complex biological samples. Enrichment
of specific glycopeptides by lectin affinity prior to MS analysis
has been widely applied to support glycopeptide identification but
was until now not available for C-mannosylated peptides. Here, we
used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated
high-mannose N-glycan binding capability, this lectin is able to retain
C- and O-mannosylated peptides. Besides testing binding abilities
to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated
peptides from complex samples of tryptic digests of HEK293 and MCF10A
whole cell extracts, which led to the identification of novel C-mannosylation
sites. In conclusion, BC2L-A enabled specific enrichment of C- and
O-mannosylated peptides and might have superior properties over other
mannose binding lectins for this purpose.