title: Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.
abstract: Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Edyta
authors.initials: E
authors.lastname: Marcon
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Harshika
authors.initials: H
authors.lastname: Jain
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Anandi
authors.initials: A
authors.lastname: Bhattacharya
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Guo
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sadhna
authors.initials: S
authors.lastname: Phanse
authors.affiliation: 1] Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. [2] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Shuye
authors.initials: S
authors.lastname: Pu
authors.affiliation: BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Byram
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland.
authors.forename: Ben C
authors.initials: BC
authors.lastname: Collins
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Dowdell
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Maria
authors.initials: M
authors.lastname: Fenner
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Guo
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ashley
authors.initials: A
authors.lastname: Hutchinson
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Kennedy
authors.affiliation: BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Brett
authors.initials: B
authors.lastname: Larsen
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Zhen-Yuan
authors.initials: ZY
authors.lastname: Lin
authors.affiliation: BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Lopez
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Loppnau
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Shane
authors.initials: S
authors.lastname: Miersch
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Tin
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Olsen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Paduch
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Mani
authors.initials: M
authors.lastname: Ravichandran
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Alma
authors.initials: A
authors.lastname: Seitova
authors.affiliation: BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
authors.forename: Gouri
authors.initials: G
authors.lastname: Vadali
authors.affiliation: BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
authors.forename: Maryann S
authors.initials: MS
authors.lastname: Vogelsang
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Whiteaker
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Zhong
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhong
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: 1] Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland. [3] Faculty of Science, University of Zurich, Zurich, Switzerland.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.affiliation: 1] Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Andrew
authors.identifier: 000000018995246X
authors.initials: A
authors.lastname: Emili
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Lori
authors.initials: L
authors.lastname: Frappier
authors.affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.affiliation: 1] Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Gstaiger
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Paulovich
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Shoshana J
authors.initials: SJ
authors.lastname: Wodak
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
authors.affiliation: 1] Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Aled M
authors.initials: AM
authors.lastname: Edwards
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D002843:Chromatin / Q000737:chemistry*; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D002843:Chromatin; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D011506:Proteins; D020543:Proteome