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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: MoP-274
Session: Supramolecular Complexes
Presentation date: Mon, Aug 28, 2006
Presentation time: 14:30 – 16:00

Detection of Intact Non-covalent Complexes in Complex Biological Mixtures by Cryodetection MALDI-TOF Mass Spectrometry

Oscar Yanes1, Alexis Nazabal2, Ryan Wenzel2, Francesc Xavier Aviles1, Renato Zenobi2

1 Institut de Biotecnologia i Biomedicina, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain
2 Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Tech, Zurich, Switzerland

Correspondence address: Oscar Yanes, Institut de Biotecnologia i Biomedicina, Universitat Autonoma de Barcelona, Protein Engineering and Proteomics, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Barcelona, 08193 Spain.

Keywords: Complex, Non-Covalent; Cross-linking; Detector, High Mass; Ionization, Matrix-Assisted Laser Desorption.

Novel aspect: Cryodetection MALDI mass spectrometry in the study of non-covalent interactions.

 

Historically, the low contribution of MALDI mass spectrometry to study intact non-covalent biomolecular complexes in solution has been linked up to the dissociation of the partners and the formation of non-specific aggregates. Here, we present a new strategy to detect intact non-covalent complexes based on cryodetection Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI ToF MS) both combined with chemical cross-linking reagents and without them. For this purpose, we have focused on the study of model interactions between the human carboxypeptidase A (hCPA) and three protease inhibitors (i.e. PCI, TCI and LCI) presented in complex mixtures of non-binding molecules derived from a biological source. The natural extract of the sea anemone Stichodactyla helianthus was also used without previous fractionation to detect the specific complex between trypsin and the endogenous SphI-1 inhibitor. Our results gain insight into the mechanism of the 'Intensity Fading MS' approach, and demonstrate that the specificity of binding is greatly favoured working in the subpicomol range, avoiding formation of non-specific aggregates both during the MALDI process or the reaction with cross-linker reagents. Many applications in the interactomic field will take advantage of new technology like cryodetection, and standard MALDI detectors may be replace in the next future.