Surface-Assisted Affinity Purification Mass Spectrometry (SAAP-MS) for Determination of Enzyme Activities
- Author(s): Yang, Hyojik
- Advisor(s): Cheng, Quan
- et al.
The development of effective assays to determine enzyme activity is fundamental to biochemical research and has broad industrial applications including high-throughput screening (HTS). In this Thesis, we have focused our effort on the development of new enzymatic assays using novel surface chemistry that enables selective capture of analytes and label-free technologies for characterization and quantification. Both covalent and non-covalent capture methods for selective enrichment of target analytes have been employed in this research. The binding process between the target analytes and the substrate surface was monitored in real time with Surface Plasmon Resonance (SPR) spectroscopy, and further confirmed and quantified with Matrix-Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS).
In the first part of this work, the non-covalent capture method was developed and employed to determine enzyme activities for two glyco-enzymes. First, α-glucosidase (α-GD) activity was studied using maltose as a substrate. A new carbohydrate probe has been synthesized and attached to a perfluorodecanethiol (PFDT)-covered surface via van der Waals interactions in a microarray format, followed by characterization by MALDI-MS. Using a ratiometric approach, we have determined the half maximal inhibitory concentration (IC50) values of inhibitors such as acarbose and epigallocatechin gallate on α-GD. In addition, N-acetylglucosamine-perfluorodecane probes have been synthesized and immobilized on PFDT-covered surface for β-1, 4-galactosyltransferase (β-GT). The design enables desalting and immobilization of carbohydrate-conjugated probes to be completed in one step. The simplicity of this approach is a high advantage for the quantification of enzymatic activity.
In the second part of this work, we have developed a robust and facile method for the fabrication of functional biochips with gold thin films. Using surface modification by self-assembled monolayers (SAMs) of lipoic acid derivatives, we have performed enzymatic reactions using droplet deposition method. The functional surface is primarily characterized by Self-Assembled Monolayers Desorption/Ionization-Mass Spectrometry (SAMDI-MS). Lipoic amido (LA)-undecaethylene glycol (PEG11)-maleimide (MAL) has been used as a model ligand to determine the binding strength between the ligand and the surface with SPR spectroscopy. Measurement of the molar mass of ligands on the surface was performed using SAMDI-MS. The maleimide moiety in the ligands allowed facile immobilization of various molecules on the surface. In addition, lipoic amido (LA)-octaethylene glycol (PEG8)-2, 3, 5, 6-tetrafluorophenol (TFP) was characterized for surface modification of the ligands by SAMDI-MS. These labeled biomolecules are then used as substrates for measurement of enzyme activities by SAMDI-MS. From the results, we have shown that LA derivatives bind effectively onto gold surfaces, providing a simple platform for surface modification of various biomolecules and as substrates for MALDI-based enzymatic assays.