Books
Chapters in books
7. Cell surface biotinylation using furan cross-linking chemistry
Maria Fernandez Fernandez (UGent) , Laia Miret Casals (UGent) , Annemieke Madder (UGent) and Kris Gevaert (UGent)
A detailed study of the cellular surfaceome poses major challenges for mass spectrometry analysis. Surface proteins are low abundant compared to intracellular proteins, and their inefficient extraction in aqueous medium leads to their aggregation and precipitation. To tackle such problems, surface biotinylation is frequently used to tag surface proteins with biotin, allowing for their enrichment, leading to a more sensitive mapping of surface proteomes. We here detail a new surface biotinylation protocol based on furan-biotin affinity purification to enrich plasma membrane proteins for proteomics. This protocol involves biotinylation of cell surface membrane proteins on viable cells, followed by affinity enrichment using streptavidin beads, trypsin digestion, peptide cleanup, and LC-MS/MS analysis.
6. Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions
Marleen Van Troys, Willem Vannecke, Christophe Ampe and Annemieke Madder
Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the oxidation of the furan moiety, which can be achieved by either addition of an external oxidation signal or by the reactive oxygen species produced by the cell. The cross-linked ligand-GPCR complex is subsequently detected by Western blotting based on the biotin label that is incorporated in the peptide ligand.
5. Synthetic peptides for DNA recognition inspired by transcription factors.
Abhishek Iyer, Yara Ruiz-Garcia and Annemieke Madder
Transcription Factors (TFs) are key players in the conversion of the information held in mRNA into a ’language’ that the body can understand. This they achieve by specifically recognising and binding to short regulatory DNA sequences which control the gene. These interactions between DNA and proteins are extremely specific and of very high affinity. Consequently, much effort has been devoted to elucidating TF structures and their modes of binding to DNA. It is therefore not surprising that TFs have been the main role models for the design and synthesis of DNA-binding peptides. The current chapter, after giving an overview of known TF structures, describes the different peptide-based DNA binding systems that have been conceived and studied, ranging from replacement of the TF dimerization domain by non-peptide scaffolds, through the connection of major-groove-recognising entities to minor-groove binders, to the further miniaturisation of the systems towards monomeric peptide sequences still capable of binding to nucleic acid duplexes. Thanks to these miniaturisation efforts, the systems have finally reached a stage where therapeutic applications can be envisaged.
4. Synthetic Protocol for AFCS: a Biologically Active Fluorescent Castasterone Analog Conjugated to an Alexa Fluor 647 Dye.
Johan M. Winne, Niloufer G. Irani, Jos Van den Begin, and Annemieke Madder
Synthetic derivatization of hormonally active brassinosteroids (BRs) can provide useful small molecule tools to probe BR signaling pathways, such as fluorescent analogs. However, most biologically active BRs are not suitable for direct chemical conjugation techniques because their derivatization typically requires extensive synthetic work and chemistry expertise. Here, we describe an operationally simple, two-step procedure to prepare and purify an Alexa Fluor 647-castasterone (AFCS) from commercially available materials. The reported strategy is also amenable to the introduction of various other amine-based labeling groups.
3. Site-selective peptide and protein labelling and crosslinking.
E. Antonatou, S. B. Gunnoo, W. Vannecke and A. Madder
Peptide and protein labelling methods have been under continuous development over the last few decades. Labelled proteins and peptides are undoubtedly indispensable for the study of biological processes at a molecular level. Nowadays, labels can be obtained via a plethora of different routes using native as well as non-native functionalities present or deliberately incorporated into the biomolecule of interest. Often, peptide ligand–protein interactions or multimeric protein complexes are involved in biological cascades of many different types and the development of efficient and site-selective crosslinking methods for freezing those transient interactions has been essential in furthering our understanding of the intricate details of cell-based machineries. Given the event of next generation, biologicals-based drug design, this research and its applications have now even reached the pharmaceutical development stage with antibody–drug conjugates (often referred to as ADCs) at the forefront of modern targeted delivery approaches. In the current chapter, we aim to provide a comprehensive overview of the most important strategies towards the generation of proteins and peptides modified in a genuine site-selective (as opposed to amino-acid selective) way as well as the application of some of those and alternative methodologies in the generation of site-specifically and covalently crosslinked peptide ligand–protein and protein–protein complexes.
(2017) Royal Society of Chemistry, Amino Acids, Peptides and Proteins. vol. 41. p.53-99, Maxim Ryadnov & Ferenc Hudecz (Eds.)
2. Interstrand Cross-Linking of Nucleic Acids: From History to Recent and Future Applications.
Ellen Gyssels, Nathalie De Laet, Emily Lumley and Annemieke Madder
This chapter contains an elaborate overview of various methods that have been developed for interstrand cross-linking of nucleic acids. The existing cross-link methodologies can be subcategorized in different groups, each interesting depending on the requirements of the envisaged application. An endogenous or exogenous bifunctional compound can react with two different nucleophilic groups on the nucleobases, resulting in fast and high-yielding cross-linking. However, when site selectivity of the formed cross-link is desired, other approaches are required. Therefore, a series of methodologies are at hand where functionalities are introduced in oligonucleotide probes, which can be intrinsically reactive or can be triggered and activated at a selected time upon target recognition. In a third class, both strands are modified with orthogonal groups. After reaction of the introduced functionalities, a cross-link is formed at a specific and fixed position. The chapter ends with an overview of and outlook to present and future applications. It is thus shown that cross-linking agents can be exploited as useful therapeutic or diagnostic tools and have further proven their utility in the stabilization or structure elucidation of cross-linked systems aimed at studying and understanding the repair systems in a cell.
1. DNA Interstrand Cross-link Formation by using Furan as a Masked Reactive Aldehyde.
Lieselot L.G. Carrette, Ellen Gyssels, Annemieke Madder
This unit describes a method for interstrand cross-linking between a furan-modified oligonucleotide and its unmodified complement. The synthesis of two furan-modified phosphoramidites, selected based on high cross-linking yield versus improved cross-linking selectivity, is described. The methods allow gram-scale synthesis starting from stable and readily available furan derivatives. Cross-linking requires selective oxidation of the furan moiety to an aldehyde. The masked nature of the latter avoids undesired and off-target reactions, resulting in clean and high-yield cross-link formation.