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Weak Ligand-Protein Interactions Studied Using Electrospray Ionization Mass Spectrometry
- Author / Creator
- Baez Bolivar, Erick G.
This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) based methods for the direct quantification and characterization of low affinity interactions between carbohydrates (glycans) and glycan binding proteins (GBPs) using nanoflow ESI emitters, from now on submicron emitters.
The affinities of most monovalent glycan‒GBP complexes are typically weak (dissociation constant (Kd) >M) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how ESI-MS, implemented with submicron emitters with inner diameters of ~50 nm, allows for the facile quantification of low affinity glycan-GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of non-specific glycan‒GBP binding (false positives) during the ESI process up to ~mM glycan concentrations. Thus, interactions with affinities as low as ~5 mM can be measured directly from the mass spectrum. The general suppression of non-specific adducts (including non-volatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium dependent manner and are important regulators of immune response. At physiologically-relevant calcium ion concentrations (2 mM ‒ 3 mM), the extent of Ca2+ non-specific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Moreover, we demonstrated how the use of submicron emitters and suppression of non-specific binding enable the quantification of labile (prone to in-source dissociation) glycan‒GBP interactions.
On the other hand, the implementation of an enzyme-aided ESI-MS-based strategy for the quantification of α2,3-Neu5Ac on PSA is described. The strategy takes advantage of time-resolved data collection by ESI-MS to calculate the relative abundances of asialo-PSA, monosialo-PSA and PSA during the reaction period. When reaction progress reaches a plateau, the relative abundance of asialo-PSA yields information about the content of α2,3-Neu5Ac-linked to PSA that was initially incorporated on the glycoprotein. Neuraminidase S (NeuS) was demonstrated to be an enzyme that specifically cleaves α2,3-linked Neu5Ac. The reliability of the method for the quantification of α2,3-Neu5Ac-linked to PSA was validated using an internal standard (Neu5Ac-13C3). Also, a purification method was developed for the extraction and concentration of PSA from blood serum. Yet, a limit of quantification (LOQ) experiment using female blood serum with no detectable PSA levels must be carried out to assess the PSA amount in real samples that this approach is capable of extracting and analyzing. This is a requirement to explore the application of this strategy for clinical purposes.
- Graduation date
- Fall 2021
- Type of Item
- Master of Science
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.