QUANTITATIVE NON-ANTIBODY AFFINITY LC-MS/MS ANALYSIS OF GLYCOPROTEINS

Significance of the Topic

Glycoproteins play critical roles as biomarkers in oncology and virology, but their heterogeneous glycosylation and low abundance present analytical challenges. Reliable, isoform-specific quantitation of glycoproteins by LC-MS/MS is essential for understanding disease mechanisms, improving diagnostics and guiding therapeutic decisions.

Objectives and Study Overview

This study evaluated non-antibody affinity reagents (Affimers and molecularly imprinted polymers) against traditional antibodies for enriching three model glycoprotein targets: carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125) and SARS-CoV-2 Spike S1. Key goals were to optimize on-bead digestion workflows, assess the impact of PNGase F-mediated deglycosylation on digestion efficiency and sensitivity, and compare enrichment performance across reagent classes.

Methodology and Instrumentation

Affinity reagents were immobilized on magnetic beads via biotin-streptavidin or covalent coupling. Following capture, proteins underwent on-bead denaturation, reduction, alkylation then single-step digestion combining PNGase F and trypsin. Released peptides were analyzed by UPLC-MS/MS in positive-ion MRM mode.

• Instrumentation: Waters ACQUITY UPLC I-Class coupled to a Xevo TQ-XS triple quadrupole with ESI source
• Workflow steps: affinity capture → on-bead sample prep → elution → MRM quantitation

Main Results and Discussion

Incorporating PNGase F increased CEA peptide signals by over five orders of magnitude and improved CA-125 detection by 10–50×. Spike S1 showed minimal benefit from deglycosylation, consistent with its low glycan content. Affimer- and antibody-based enrichment yielded comparable sensitivity for CEA, while MIP particles provided selective binding to Spike with minimal background. Some non-specific binding to supports was noted, highlighting the need for reagent-specific optimization.

Benefits and Practical Applications

• PNGase F integration enhances digestion efficiency and sensitivity in bottom-up glycoprotein assays.
• Non-antibody reagents (Affimers, MIPs) offer robust alternatives to antibodies, supporting workflow flexibility and potential cost savings.
• The combined affinity/digestion protocol is compatible with automated platforms for high-throughput biomarker quantitation.

Future Trends and Potential Applications

Advances in synthetic affinity reagents and integrated digestion strategies will drive multiplexed glycoprotein profiling in clinical diagnostics. Emerging directions include isoform-specific glycopeptide targeting, streamlined automation of on-bead workflows and expansion to additional disease markers and biofluids.

Conclusion

This work demonstrates that coupling enzymatic deglycosylation with on-bead digestion markedly improves glycoprotein detection by LC-MS/MS. Affimers and MIPs show comparable performance to antibodies, paving the way for versatile affinity platforms in quantitative glycoproteomics.

References

1. Shamsuddin SH et al. Sci Rep. 2021;11(1):744
2. Bossi A et al. Biosens Bioelectron. 2007;22(6):1131–1137
3. Van Puyvelde et al. Anal Chem. 2022;94(50):17379–17387
4. Native Antigen Company. SARS-CoV-2 Spike S1 product data
5. Waters Technologies. Xevo, ACQUITY and UPLC trademarks