Multiplexed Comparative Analysis of Intact Glycopeptides Using Electron-Transfer Dissociation and Synchronous Precursor Selection Based Triple-Stage Mass Spectrometry

A recently
developed synchronous precursor selection (SPS) mass
spectrometry to the third (MS3) protocol enables more accurate multiplexed
quantification of proteins/peptides using tandem mass tags (TMT) through
comparison of reporter ion intensities at the MS3 level. However,
challenges still exist for TMT-based simultaneous quantification and
identification of intact glycopeptides due to inefficient peptide
backbone fragmentation when using collision-induced dissociation (CID).
To overcome this limitation, here we report an improved SPS/ETD workflow
for TMT-based intact glycopeptide quantification and identification.
The SPS/ETD approach was implemented on an Orbitrap Tribrid mass spectrometer
and begins with selection of a parent ion in the MS scan, followed
by tandem mass spectrometry (MS2) fragmentation by CID in the ion
trap. Following MS2 fragmentation, SPS enables simultaneous isolation
of the top 10 MS2 fragment ions for further higher energy collisional
dissociation (HCD) fragmentation with the resulting MS3 fragments
detected in an Orbitrap analyzer. Here, in addition to the standard
SPS workflow, an electron-transfer dissociation (ETD) MS2 was performed
and analyzed in the ion trap. The resultant ETD and CID spectra were
used for the identification of the intact glycopeptides, while the
quantitative comparison of site-specific glycans was achieved utilizing
TMT reporter ions from HCD MS3 spectra. For intact glycopeptides,
through systematic optimization and evaluation using a glycoprotein
interference model, the SPS/ETD approach was demonstrated to offer
improved accuracy, precision, and sensitivity compared to traditional
data-dependent MS2 quantification, while maintaining the glycopeptide
identification capability. Finally, this workflow was applied for
the site-specific quantitative comparison of the glycoforms for two
therapeutic enzymes (Cerezyme and VPRIV) and their different lots.
The results demonstrate that this workflow is suitable for TMT-based
intact glycopeptide characterization of glycoproteins.