Acid deprotection of covalently immobilized peptide probes on glass slides for peptide microarrays.
Protein microarray technology has shown great advancements in the field of biomedical research and diagnosis, it allows to study and understand protein activities and protein - ligand interactions (e.g. detection of antigen-autoantibody interaction in autoimmune diseases. Autoantibodies are frequently targeted against antigens of the cell nucleus (double and single stranded DNA, histones, and nuclear antigens). The biological activities of proteins (e.g. enzymes, antibodies...) are controlled by peptides sequences of the active site. Consequently, we were interested in the investigation of peptide microarrays in order to further implement in situ peptide synthesis, in particular, deprotection reaction on glass supported peptides. In this work, a protected and biotinylated synthetic peptide was covalently immobilized onto amino functionalized glass surface by activation of its the C-terminus; this allows to orientate the peptide onto the surface. The peptide contains a fragment of the C-terminal end of the human histone H3 protein. The immobilized peptide was then deprotected by using concentrated trifluoroacetic acid solution. After the deprotection, surface stability and peptide grafting density were evaluated by indirect labelling of the immobilized peptide using Cy3 streptavidin conjugates. We also studied biological interaction of IgG polyclonal anti-histone H3 antibody with the immobilized peptide epitope to insure the efficiency of the acid deprotection. The specificity of the antibody interaction with the protected versus non protected peptides. This approach may be applied to in situ synthetic and prototected peptides, in order to elaborate a micro-immunoassay prototype for measurement of peptide-protein interactions on high density microarrays, and detection of antibodies in biological fluids such as serum.
El Khoury
Graziella
G
Lyon Nanotechnology Institute, Ecole centrale de Lyon, Ecully 69134 France. Graziella.El-Khoury@ec-lyon.fr
Laurenceau
Emmanuelle
E
Dugas
Vincent
V
Chevolot
Yann
Y
Mérieux
Yves
Y
Duclos
Marie-Christine
MC
Souteyrand
Eliane
E
Rigal
Dominique
D
Wallach
Jean
J
Cloarec
Jean Pierre
JP
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004867:Equipment Design; D005898:Glass; D006657:Histones / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation*; D011506:Proteins / Q000737:chemistry*
D000906:Antibodies; D006657:Histones; D007074:Immunoglobulin G; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011506:Proteins