title: Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry.
abstract: Protein phosphorylation is a ubiquitous post-translational modification that affects a significant subset of the proteome and plays an especially important role in signal transduction and cell cycle control in eukaryotic organisms. Recently developed methods that couple multidimensional liquid chromatography to electrospray mass spectrometers can be used to analyze entire phosphoproteomes. However, they require considerable investments and technical skills that are only available in a few highly specialized laboratories. These methods also appear to be biased. Statistical analyses show that peptides from abundant proteins and multiply phosphorylated peptides are disproportionately identified. We describe an economic alternative that utilizes a phospho-affinity step to isolate the intact phosphoproteins. These are subsequently characterized by electrophoresis and identified by direct de novo sequencing using tandem mass spectrometry. We applied this technique to probe signal-induced changes in the phosphoproteome of human U937 cells, and found that the pools of two cancer-related phosphoproteins implicated in intracellular hormones signaling are dramatically altered in the course of monocyte to macrophage differentiation.
authors.affiliation: Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, IL 60607, USA. metodi@uic.edu
authors.forename: Metodi V
authors.initials: MV
authors.lastname: Metodiev
authors.forename: Anna
authors.initials: A
authors.lastname: Timanova
authors.forename: David E
authors.initials: DE
authors.lastname: Stone
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002457:Cell Extracts / Q000737:chemistry; D002498:Centrifugation; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects; D009363:Neoplasm Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000191:economics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate