Fundamental studies on structural proteomics: photoactivatable cross-linkers and conformational changes probed by ion mobility mass spectrometry

Author(s)

Alexandre Ferreira Gomes

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2010-07-30

Examining board members

Fábio Cesar Gozzo; Emer Suavinho Ferro; Marcos Nogueira Eberlin

• Advisor: Fábio Cesar Gozzo

Abstract

The use of mass spectrometry (MS) in proteomics has evolved extensively, up to the point where it became routinely employed in protein sequencing and determination of post-translational modifications. However, the use of MS in structural studies of proteins is limited, despite the many advantages it could bring to the area. In this sense, chemical cross-linking coupled to mass spectrometry for structural studies of proteins aims to obtain structural information in terms of spatial distance restrictions, determined by intra- and intermolecular cross-links between side chains of amino acid residues. This is achieved by reaction of the target protein system with bifunctional compounds, followed by proteolysis and analysis of the modified peptides by MS. The first part of this work describes the application of a novel, heterobifunctional and cleavable cross-linker, containing a diazirine photoactivatable group, for cross-linking of peptides and proteins. Reaction with this cross-linker yielded unique cross-links for peptides and proteins, and such modified peptides where characterized by MS/MS, allowing the discovery of a marker ion, that may be used to identify cross-linked peptides. The second part of the work focused on the application of traveling-wave ion mobility (TWIM) coupled to MS for a comparative analysis of protein ions both before and after cross-linking reactions, aiming to evaluate the effect of these modifications on protein structures in terms of variation of collision cross sections (CCS) of their ions. TWIM-MS analysis of these systems demonstrated the presence of compact gas phase conformations of lower CCS after the cross-linking reaction and formation of intramolecular cross-links, indicating that chemical cross-linking restricts the structural dynamics of gas phase protein ions. To validate this method, experimental CCS values were then compared with theoretical values obtained computationally for available crystallographic or NMR structures. (AU)

• FAPESP's process: 08/02043-0 - Structural and Conformational Studies of Proteins and Peptides Using Cross-linking and Ion Mobility Coupled To Mass Spectrometry
• Grantee: Alexandre Ferreira Gomes
• Support Opportunities: Scholarships in Brazil - Master