Immunoaffinity isolation of modified peptides from complex...

C - Chemistry – Metallurgy – 07 – K

Patent

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C07K 1/14 (2006.01) C07K 1/22 (2006.01) C07K 16/18 (2006.01) C07K 16/40 (2006.01) C07K 16/44 (2006.01) G01N 30/72 (2006.01) G01N 33/53 (2006.01) G01N 33/543 (2006.01) G01N 33/551 (2006.01) G01N 33/68 (2006.01)

Patent

CA 2454605

The invention provides methods for isolating a modified peptide from a complex mixture of peptides, the method comprising the steps of: (a) obtaining a proteinaceous preparation from an organism, wherein the preparation comprises modified peptides from two or more different proteins; (b) contacting the preparation with at least one immobilized modification-specific antibody; and (c) isolating at least one modified peptide specifically bound by the immobilized modification-specific antibody in step (b). The method may further comprise the step of (d) characterizing the modified peptide isolated in step (c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS), and/or MS3 analysis, or the step of (e) utilizing a search program to substantially match the spectra obtained for the modified peptide during the characterization of step (d) with the spectra for a known peptide sequence, thereby identifying the parent protein(s) of the modified peptide. Also provided are an immunoaffinity isolation device comprising a modification-specific antibody, and antibodies against novel UFD1 and PTN6 phosphorylation sites.

L'invention concerne des méthodes d'isolation d'un peptide modifié à partir d'un mélange complexe de peptides. Une méthode consiste à (a) obtenir une préparation protéique à partir d'un organisme, ladite préparation renfermant des peptides modifiés provenant d'au moins deux protéines différentes, (b) mettre en contact la préparation avec au moins un anticorps spécifique de modification immobilisée, et (c) isoler au moins un peptide modifié spécifiquement lié par ledit anticorps de l'étape (b). Ce procédé consiste ensuite, à (d) caractériser le peptide modifié isolé à l'étape (c) par spectrométrie de masse, spectrométrie de masse en tandem et/ou une analyse MS?3¿ ou (e) à utiliser un programme de recherche pour faire correspondre les spectres obtenus pour le peptide modifié au cours de la caractérisation de l'étape (d) avec les spectres d'une séquence de peptides connue, ce qui permet d'identifier la protéine parente du peptide modifié. Ladite invention a aussi trait à un dispositif d'isolation par immunoaffinité comprenant un anticorps spécifique de modification et des anticorps contre de nouveaux sites de phosphorylation UFD1 et PTN6.

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