Combination of solid-phase affinity capture on magnetic beads and mass spectrometry to study non-covalent interactions: example of minor groove binding drugs.
A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation.
Schlosser
Gitta
G
Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., via Roma 52 a/c, 83100 Avellino, Italy.
Vékey
Károly
K
Malorni
Antonio
A
Pocsfalvi
Gabriella
G
D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D008968:Molecular Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D004364:Pharmaceutical Preparations; D001710:Biotin; D019809:Streptavidin