Strategic Applications of Negative-Mode LC-MS/MS Analyses to Expedite Confident Mass Spectrometry-Based Identification of Multiple Glycosylated Peptides

Although
recent advances in mass spectrometry (MS) have enabled
meaningful glycoproteomic undertakings, many technical limitations
remain unsolved. Among these, the ability to efficiently sequence
the peptide backbone for de novo identification,
delineating multiple N- and O-glycosylation
sites on single glycopeptides, and deriving more glycan structure
information to discriminate isomeric glycoforms are well acknowledged
practical problems to be tackled. To address these issues, we explored
the use of negative-mode MS2/MS3 fragmentation
to supplement current nanoLC-MS2-based sequencing and identification
of intact glycopeptides largely performed in positive mode. Consistent
with previous reports by others, we found that sulfation and sialylation
drastically alter the MS2 fragmentation pattern of glycopeptides
in negative mode and the characteristic features identified can be
utilized to program the most informative MS3 on the glycan
moiety itself. Importantly, direct elimination of one or more O-glycans under negative-mode MS2 affords an
easy way to discover additional O-glycosylations
on a multiply glycosylated peptide by virtue of enumerating the dehydration
scars imprinted on the O-glycosylated sites. Moreover,
the characteristic peptide core ion carrying a ring cleavage remnant
of the innermost amino sugar residue of an N-glycan
can be relied upon to filter out all related N-glycopeptides
carrying additional O-glycans defined by specific
mass increments. Such enhanced ability to advance from definitive
identification of single to multiple site-specific glycosylation on
the same peptide backbones is anticipated to have a significant impact
on the level of structural and biological insights one can gain in
glycoproteomic applications.