title: A new approach for quantitative phosphoproteomic dissection of signaling pathways applied to T cell receptor activation.
abstract: Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.
authors: Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
authors: Vinh
authors: V
authors: Nguyen
authors: Lulu
authors: L
authors: Cao
authors: Jonathan T
authors: JT
authors: Lin
authors: Norris
authors: N
authors: Hung
authors: Anna
authors: A
authors: Ritz
authors: Kebing
authors: K
authors: Yu
authors: Radu
authors: R
authors: Jianu
authors: Samuel P
authors: SP
authors: Ulin
authors: Benjamin J
authors: BJ
authors: Raphael
authors: David H
authors: DH
authors: Laidlaw
authors: Laurent
authors: L
authors: Brossay
authors: Arthur R
authors: AR
authors: Salomon
mesh_terms: D001665:Binding Sites; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000737:chemistry
chemical_list: D010455:Peptides; D010748:Phosphopeptides; D020543:Proteome; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human