Fast NMR-Based Assessment of Cancer-Associated Protein Glycosylations from Serum Samples

Nuclear magnetic resonance (NMR) spectra of blood serum
and plasma
show signals arising from metabolites, lipoproteins, and N-acetyl methyl groups of N-glycans covalently linked
to acute-phase proteins. These glycan signals often called glycoprotein
A (GlycA) and glycoprotein B (GlycB) arise from N-acetyl methyl groups and have been proposed as biomarkers, initially
for cardiovascular diseases, but also for other inflammatory conditions.
For the detection of glycan resonances, J-edited,
diffusion, and relaxation filtered NMR spectroscopy (JEDI) has been
proposed to suppress the lipoprotein signals. JEDI is however limited
to measure those acetyl signals, whereas all other glycan resonance
cannot be observed. For improved glycoprotein profiling, the signals
arising from the pyranose ring protons are essential. Here, we show
how selective frequency excitation combined with scalar coupling filtering
can be used to dramatically increase the number of N-glycan signals observable in NMR spectra of serum and plasma samples,
facilitating glycosylation profiling in less than 30 min. This approach
grants selective detection of sialylation, galactosylation, N-acetylglucosaminylation, and fucosylation of dominant N-glycans and, to some extent, N-glycan
branching complexity. Notably, sialylated and nonsialylated Lewisx and Lewisa antigens can also be observed. Lewisa antigen is well established as a cancer biomarker, known
as CA19-9. NMR glycosylation profiles from nine isolated serum glycoproteins
show excellent agreement with well-established UHPLC-MS analysis.
The proposed NMR method facilitates the detection of glycoprotein
biomarkers without the need for enzymatic treatment of serum or plasma
and provides a robust read-out as exemplified by samples from 33 patients
with hepatocellular carcinoma.