title: Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins.
abstract: Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, P. R. China.
authors.forename: Guochen
authors.initials: G
authors.lastname: Qin
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, P. R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, P. R. China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Chen
authors.affiliation: Shanghai Chenshan Plant Science Research Center and Shanghai Chenshan Botanic Garden, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, P. R. China.
authors.forename: Zhaoqing
authors.initials: Z
authors.lastname: Chu
authors.affiliation: Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, P. R. China.|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, K1A 0K9, Canada.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D007553:Isotope Labeling; D008745:Methylation; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000378:metabolism; D060066:Protein Interaction Maps; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D029681:Arabidopsis Proteins; D010455:Peptides; D021122:Protein Subunits; C538841:RNA polymerase V, Arabidopsis; D012321:DNA-Directed RNA Polymerases