RESUMO
Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of â¼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
Assuntos
Proteoma/análise , Proteômica/métodos , Ubiquitinação , Aminoácidos/metabolismo , Anticorpos/química , Anticorpos/imunologia , Sítios de Ligação , Cromatografia Líquida/métodos , Reagentes de Ligações Cruzadas/química , Inibidores de Cisteína Proteinase/farmacologia , Glicilglicina/imunologia , Humanos , Marcação por Isótopo/métodos , Células Jurkat , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Proteínas Ubiquitinadas/análise , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismoRESUMO
Nuclear magnetic resonance has been used to study the structure of the anti-spin label antibody AN02 combining site and kinetic rates for the hapten-antibody reaction. The association reaction for the hapten dinitrophenyl-diglycine (DNP-diGly) is diffusion-limited. The activation enthalpy for association, 5.1 kcal/mol, is close to the activation enthalpy for diffusion in water. Several reliable resonance assignments have been made with the aid of recently reported crystal structure. Structural data deduced from the nuclear magnetic resonance (n.m.r.) spectra compare favorably with the crystal structure in terms of the combining site amino acid composition, distances of tyrosine residues from the unpaired electron of the hapten, and residues in direct contact with the hapten. Evidence is presented that a single binding site region tyrosine residue can assume two distinct conformations on binding of DNP-diGly. The AN02 antibody is an autoantibody. Dimerization of the Fab fragments is blocked by the hapten DNP-diGly. The n.m.r. spectra suggests that some of the amino acid residues involved in the binding of the DNP-hapten are also involved in the Fab dimerization.
