title: Analysis of the subcellular phosphoproteome using a novel phosphoproteomic reactor.
abstract: Protein phosphorylation is an important post-translational modification involved in the regulation of many cellular processes. Mass spectrometry has been successfully used to identify protein phosphorylation in specific pathways and for global phosphoproteomic analysis. However, phosphoproteomics approaches do not evaluate the subcellular localization of the phosphorylated forms of proteins, which is an important factor for understanding the roles of protein phosphorylation on a global scale. The in-depth mapping of protein phosphorylation at the subcellular level necessitates the development of new methods capable of specifically and efficiently enriching phosphopeptides from highly complex samples. Here, we report a novel microfluidic device called the phosphoproteomic reactor that combines efficient processing of proteins followed by phosphopeptide enrichment by Ti-IMAC. To illustrate the potential of this novel technology, we mapped the phosphoproteins in subcellular organelles of liver cells. Fifteen subcellular fractions from liver cell cultures were processed on the phosphoproteomic reactor in combination with nano-LC-MS/MS analysis. We identified thousands of phosphorylation sites in over 600 phosphoproteins in different organelles using minute amounts of starting material. Overall, this approach provides a new avenue for studying the phosphoproteome of the subcellular organelles.
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada.
authors.forename: Houjiang
authors.initials: H
authors.lastname: Zhou
authors.forename: Fred
authors.initials: F
authors.lastname: Elisma
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Denis
authors.forename: Theodore G
authors.initials: TG
authors.lastname: Wright
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.forename: Weimin
authors.initials: W
authors.lastname: Hou
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D016000:Cluster Analysis; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation*; D015388:Organelles / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D020543:Proteome