title: Identification of a novel mitochondrial interacting protein of C1QBP using subcellular fractionation coupled with CoIP-MS.
abstract: The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China. chenruibing@tijmu.edu.cn.
authors.forename: Ruibing
authors.initials: R
authors.lastname: Chen
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Xiao
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Huajun
authors.initials: H
authors.lastname: Gao
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Yajing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Li
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China.
authors.forename: Yunde
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy; Research Center of Basic Medical Sciences; School of Medical Laboratory, Tianjin Medical University, Tianjin, 300070, China. zhangning@tijmu.edu.cn.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
mesh_terms: D001324:Autoantigens / Q000032:analysis / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001324:Autoantigens; C087299:C1QBP protein, human; D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D011768:Pyruvate Dehydrogenase Complex; C052359:DLAT protein, human; D051047:Dihydrolipoyllysine-Residue Acetyltransferase
keywords: C1QBP; CoIP-MS/MS; LC-MS/MS; Mitochondria; Protein-protein interaction; Subcellular fractionation