title: Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.
abstract: Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies.
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, Canada.
authors.forename: Jiefei
authors.initials: J
authors.lastname: Tong
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, P. R. China.
authors.forename: Biyin
authors.initials: B
authors.lastname: Cao
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Canada.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Martyn
authors.affiliation: SPARC BioCentre, Hospital for Sick Children, Toronto, Canada.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Krieger
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, Canada.|SPARC BioCentre, Hospital for Sick Children, Toronto, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Canada.|Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Canada.
authors.forename: Bradley
authors.initials: B
authors.lastname: Yates
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Canada.|Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: Department of Biochemistry and Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada.
authors.forename: Shawn S C
authors.initials: SS
authors.lastname: Li
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, P. R. China.
authors.forename: Xinliang
authors.initials: X
authors.lastname: Mao
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Canada.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D018909:src Homology Domains
chemical_list: D010455:Peptides; D020543:Proteome; D019000:Phosphotyrosine
keywords: Affinity purification; Anti-phosphotyrosine; EGF receptor; HeLa; Phospho-proteomics; Phosphotyrosine; SH2 domain; Superbinder; Technology