Capillary-Scale Frontal Affinity Chromatography/MALDI Tandem Mass Spectrometry Using Protein-Doped Monolithic Silica Columns

Abstract

Frontal affinity chromatography (FAC) interfaced with electrospray mass spectrometry (ESI-MS) has been reported as a potential method for screening of compound mixtures against immobilized target proteins. However, the interfacing of bioaffinity columns to ESI-MS requires that the eluent that passes through the protein-loaded column have a relatively low ionic strength to produce a stable spray. Such low ionic strength solvents can cause serious problems with protein stability and may also affect binding constants and lead to high nonspecific binding to the column. Herein, we report on the interfacing of bioaffinity columns to matrix-assisted laser desorption/ionization (MALDI) MS/MS as a new platform for FAC/MS studies. Capillary columns containing a monolithic silica material with entrapped dihydrofolate reductase were used for frontal affinity chromatography of small-molecule mixtures. The output from the column was combined with a second stream containing alpha-cyano-hydoxycinnamic acid in methanol and was deposited using a nebulizer-assisted electrospray method onto a conventional MALDI plate that moved relative to the column via a computer-controlled x-y stage, creating a semipermanent record of the FAC run. The use of MALDI MS/MS allowed for buffers with significantly higher ionic strength to be used for FAC studies, which reduced nonspecific binding of ionic compounds and allowed for better retention of protein activity over multiple runs. Following deposition, MALDI analysis required only a fraction of the chromatographic run time, and the deposited track could be rerun multiple times to optimize ionization parameters and allow signal averaging to improve the signal-to-noise ratio. Furthermore, high levels of potential inhibitors could be detected via MALDI with limited ion suppression effects. Both MALDI- and ESI-based analysis showed similar retention of inhibitors present in compound mixtures when using identical ionic strength conditions. The results show that FAC/MALDI-MS should provide advantages over FAC/ESI-MS for high-throughput screening of compound mixtures.

New monolithic chromatographic supports for macromolecules immobilization: Challenges and opportunities

“…The tendency of DHFR to denature in low ionic strength running buffer required for ESI-MS (2 mM ammomiun acetate buffer) has stimulated the integration of the developed monolithic bioaffinity columns with MALDI-MS/MS detection [31]. The interfacing involves mixing the column effluent with a suitable matrix followed by continuous nebulizer-assisted electrospray deposition of the mixture onto MALDI plate.…”

Affinity monolith chromatography

“…The entrapped protein was found to have good activity for some inhibitors of DHFR (e. g., trimethoprim and pyrimethamine) and was used with MS to determine the binding constants of DHFR for pyrimethamine. A similar column was interfaced with MALDI MS/MS [67]. Other ligands that have been studied using sol-gel entrapment and capillary columns include nicotinic acetylcholine receptor from Torpedo californica, dopamine D2Short receptor [62,63,69], protein tyrosine kinase [64], firefly luciferase [65], aptamers [68], and antifluorescein antibodies [66].…”

Integration of Biomolecules into analytical Systems by Means of Silica Sol-Gel Technology

“…68 Most applications of this technique use monolithic columns fabricated in a capillary to simplify the procedures, and involve the use of a capillary LC (or nano-LC) system. [69][70][71] The column is prepared from a mixture of DGS; PEG, which controls morphology; APTES, which provides cationic sites that counterbalance the anionic charge of the silica to reduce nonselective interaction; and a buffered solution of the proteins to provide bioaffinity sites within the column. The resulting silica has a bimodal pore distribution with macropores (>0.1 μm) that allow good flow of eluent with minimal back pressure, and mesopores (~3 -5 nm) that retain a significant fraction of the entrapped protein.…”

“…70,72 The second method for immobilization of biomolecules on bimodal monolithic supports for LC involves an activated silica monolithic column on which biomolecules are immobilized. For example, Calleri et al used a thoroughly dried commercial monolithic silica column (4.6 mm i.d.)…”