Capillary-scale frontal affinity chromatography/MALDI tandem mass spectrometry using protein-doped monolithic silica columns.
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.
Kovarik
Peter
P
MDS/Sciex, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada.
Hodgson
Richard J
RJ
Covey
Tom
T
Brook
Michael A
MA
Brennan
John D
JD
D001331:Automation; D001665:Binding Sites; D002021:Buffers; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003373:Coumaric Acids / Q000737:chemistry; D019793:Fluorescein / Q000032:analysis / Q000737:chemistry; D005492:Folic Acid / Q000032:analysis / Q000737:chemistry; D007090:Image Interpretation, Computer-Assisted; D009994:Osmolar Concentration; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011739:Pyrimethamine / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry; D013997:Time Factors; D014295:Trimethoprim / Q000032:analysis / Q000737:chemistry
D002021:Buffers; D003373:Coumaric Acids; D011506:Proteins; D012997:Solvents; C007175:alpha-cyano-4-hydroxycinnamate; D012822:Silicon Dioxide; D005492:Folic Acid; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D019793:Fluorescein; D011739:Pyrimethamine