Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics

Deep characterization
of biologically relevant glycans remains
challenging. Porous graphitized carbon–liquid chromatography
tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation
of glycan fine structures. However, the early PGC-LC elution of smaller
glycans (tri-, tetra-, and pentasaccharides) at low organic solvent
content hampers their detection. In efforts to improve the glycan
profiling sensitivity and accuracy, we present a new capillary-flow
PGC-LC-MS/MS-based configuration comprising a post-column make-up
flow (PCMF) that supplies an ion-promoting organic solvent to separated
glycans prior to their detection by MS. The analytical performance
of this setup was systematically evaluated against our existing capillary-flow
PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the
ion intensities and signal-to-noise ratios of various classes of nonderivatized
glycans from N- and O-glycoproteins
and fructooligosaccharide mixtures were compared using methanol (MeOH)-,
isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations.
In particular, ACN- and IPA-based PCMF dramatically increased the
signal response across all glycan types (30- to 100-fold), improved
the MS/MS spectral quality, and reduced the quantitative glycoprofile
variation between replicates. In particular, the detection of the
early eluting glycans benefitted from the PCMF. The highest sensitivity
gains were achieved with the supplements of 100% ACN and IPA (equating
to 57% (v/v) net concentration at the ion source) while neither compromising
the favorable PGC-LC properties including the high peak capacity and
glycan isomer separation nor changing the MS detection behavior. In
conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics
sensitivity, coverage, and quantitative accuracy not least for the
difficult-to-detect early eluting and low-abundance glycans detached
from N- and O-glycoproteins.