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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-226
Session: Proteomics: New Methods
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Application of Affinity Proteomics and High Resolution FTICR Mass Spectrometry for Elucidation of Antibody-paratope Peptides (PAREXPROT)

Diana Klingler1, Andreas Marquardt1, Gabriela Paraschiv1, Erika R. Amstalden van Hove1,2, Marilena Manea1, Michael Przybylski1

1 University of Konstanz, Konstanz, Germany
2 Institute for Atomic and Molecular Physics AMOLF, Amsterdam, Netherlands

Correspondence address: Diana Klingler, University of Konstanz, Laboratory of Analytical Chemistry, Universitaetsstr. 10, Konstanz, Baden-Wuerttemberg, 78457 Germany.

Keywords: Affinity, Binding; Antibodies; MALDI; Mass Spectrometry, Fourier Transform.

Novel aspect: Application of affinity proteomics and high resolution FTICR mass spectrometry for elucidation of antibody-paratope peptides (PAREXPROT).

 

High resolution and high selectivity mass spectrometry has become one of the most important tools for the identification of molecular recognition structures of antigens (epitopes).1,2,3 We describe here a new approach for the identification of affinity bound proteins which has been developed in our laboratory as a key prerequisite for molecular vaccine design and targeting. For this approach a combination of immuno-affinity isolation, selective proteolytic degradation of immune complexes (paratope excision, PAREXPROT) and mass spectrometric peptide mapping was used. The direct characterisation of a paratope peptide was performed by selective binding of a specific DTT-cleavage mixture of a heavy-light chain dimer of a polyclonal anti-Lysozyme Antibody to the immobilised Lysozyme (HEL), followed by proteolytic digestion of the antigen:antibody complex (paratope excision, PAREXPROT). The paratope peptides were determined by high resolution matrix assisted laser desorption-ionisation (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry at 1-2 ppm mass accuracy. The direct identification of the paratope sequences was obtained by database search at 1-2 ppm accuracy threshold. The affinities of these paratopes were ascertained by synthesis and affinity determination of the authentic sequences.

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