title: Sensitive Approaches for the Assay of the Global Protein Tyrosine Phosphorylation in Complex Samples Using a Mutated SH2 Domain.
abstract: Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the underlying mechanisms. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng of tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop a method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS) , Dalian 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS) , Dalian 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS) , Dalian 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS) , Dalian 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.affiliation: CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS) , Dalian 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Mingliang
authors.identifier: 0000-0002-5872-9326
authors.initials: M
authors.lastname: Ye
mesh_terms: D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000032:analysis* / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
chemical_list: D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3