Chips from chips: application to the study of antibody responses to methylated proteins.
Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.
Institut de Recherche Interdisciplinaire, Villeneuve d'Ascq Cédex, France.
Gaëlle
G
Piret
Rémi
R
Desmet
Eric
E
Diesis
Hervé
H
Drobecq
Jérome
J
Segers
Carine
C
Rouanet
Anne-Sophie
AS
Debrie
Rabah
R
Boukherroub
Camille
C
Locht
Oleg
O
Melnyk
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D005260:Female; D037102:Lectins / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D051379:Mice; D008807:Mice, Inbred BALB C; D046228:Microarray Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D011506:Proteins / Q000276:immunology / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D000907:Antibodies, Bacterial; D037102:Lectins; D010455:Peptides; D011506:Proteins; C104578:heparin-binding hemagglutinin