title: Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.
abstract: Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.
authors.affiliation: Research Institute Children's Cancer Center and Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Building N63, 2nd Floor, 20251, Hamburg, Germany.
authors.forename: Sophia
authors.initials: S
authors.lastname: Buhs
authors.affiliation: Research Institute Children's Cancer Center and Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Building N63, 2nd Floor, 20251, Hamburg, Germany.
authors.forename: Helwe
authors.initials: H
authors.lastname: Gerull
authors.affiliation: Research Institute Children's Cancer Center and Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Building N63, 2nd Floor, 20251, Hamburg, Germany. nollau@uke.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Nollau
mesh_terms: D002846:Chromatography, Affinity; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011993:Recombinant Fusion Proteins; D057188:Workflow; D018909:src Homology Domains
chemical_list: D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine
keywords: Affinity purification; Cancer; Mass spectrometry; Phosphotyrosine; Protein-protein interaction; Pull down; SH2 domain; Signal transduction