Affinity Capture of a (Glyco)protein: Getting Your Sample Ready for the Mass Spectrometer
Valeriia Kuzyk(1,2), Guinevere S.M. Lageveen-Kammeijer(2), Rob Haselberg(1), Manfred Wuhrer(2), Govert W. Somsen(1) (1) Division of BioAnalytical Chemistry, Amsterdam Institute of Molecules, Medicines and Systems, Vrije Universiteit, Amsterdam, (2) Center for Proteomics and Metabolomics, Leiden University Medical Centre, Leiden
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Presenter Bio: Current research interests are focused on Glycomics and Glycoproteomics, more specifically in their application to colorectal cancer diagnostics. Educational and research background include Biochemistry, Bioinformatics and Statistics. Strongly believe in lab-on-a-chip and mass-spectrometry being the nearing future of clinical diagnostics.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction:
Extracting a pure sample of the target protein from a complex biological fluid is often crucial for reliable, repeatable and sensitive results in mass-spectrometry analysis. Special attention is to be paid to the targets with abundant PTMs, such as glycosylation. In this work we present a comparison of different affinity capturing techniques used to isolate a low-concentrated glycoprotein from biofluids, while minimizing protein background in the eluate. Alongside with that, we offer a roadmap for a capturing method development, with mapped pitfalls and points of attention.
Objectives:
Perform an evaluation study of low-concentrated protein enrichment strategies.
Methods:
This work explored the efficiency of affinity capturing methods exploiting various solid supports. Performances of sepharose-beads, amine-terminated and NHS-terminated magnetic beads, as well as monolith-immobilized gold nanoparticles were evaluated and compared in terms of capturing efficacy and background protein contamination. Key workflow steps, such as carrier vial choice, drying procedure and elution methods were determined and explored. The tests were conducted on a set of biological matrices, that differ in protein concentration and physical properties. Nanobodies (single-chain antibodies) were used in the majority of tests, showing potential as a low-cost and stable substitute of full-size monoclonal antibodies. As an extra development, we present aptamers-based approach: a promising affinity capturing ligand with unique properties and simplicity of production.
Results:
We state magnetic beads to be the preferred option for in-solution affinity capturing and highlight the value of monolithic column for background reduction in the eluate. The capturing efficacy with the method reaches 70% and the method is applicable for complex sample matrices. The resulting eluate yield is suitable for subsequent mass-spectrometry analysis.
We also present an established roadmap for affinity capturing method development.
Conclusion:
The methods we explored are valuable as a (glyco)protein enrichment step prior to mass spectrometry analysis and offer potential for exploring low-abundance protein targets in the protein-rich matrices.
= Discovery stage.
= Translation stage.
= Clinically available.
