title: Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.
abstract: We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified ∼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Bian
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.|School of Basic Medical Sciences, Qingdao University, Qingdao, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Xuguang
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kaneko
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.
authors.forename: Kai
authors.identifier: http://orcid.org/0000-0002-6045-0244
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Huadong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Courtney
authors.initials: C
authors.lastname: Voss
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Xuan
authors.initials: X
authors.lastname: Cao
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.|Department of Oncology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Litchfield
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.|Department of Oncology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.|The Children's Health Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.
authors.forename: Shawn S-C
authors.initials: SS
authors.lastname: Li
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D013058:Mass Spectrometry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D040901:Proteomics; D018909:src Homology Domains
chemical_list: D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases