Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94−101

Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.
Nature Biotechnology (Impact Factor: 41.51). 02/2005; 23(1):94-101. DOI: 10.1038/nbt1046
Source: PubMed


Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

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    • "To further address the mechanism of MERTK-mediated tyrosine phosphorylation of GDI1, HEK-293T cells were co-transfected with GDI1 constructs encoding either wild-type or mutant proteins in which known sites of GDI1 tyrosine phosphorylation were disrupted (Shisheva et al., 1999; Rush et al., 2005; Ballif et al., 2008). Western analysis of anti-phosphotyrosine immunoprecipitates showed that MERTK-induced phosphorylation of GDI1 was unaffected by mutations affecting GDI1 residues Tyr249 (GDI1 Y249F) and Tyr333 (GDI1 Y333F), but was abolished by mutation of Tyr339 (GDI1 Y339F) (Fig. 5A). "
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    ABSTRACT: Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS, but exhibited labeling patterns that were coincident in some areas and mutually exclusive in others. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015 · Experimental Eye Research
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    • "Scaffold software (v3.6.5, Proteome Software, Portland, OR) was used to display and validate MS/MS-based peptide and protein identifications. The Peptide Prophet cut off was set at 95% [45] and additionally, MS/MS spectra of all pY-peptides were individually inspected as previously described [46] to ensure high quality spectrum filtering. In addition, each phospho-peptide was assigned an Ascore [47] "
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    Full-text · Article · Jan 2015 · Proteomics
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    • "Phosphoproteome is usually analyzed by coupling an enrichment strategy with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A number of phosphopeptide enrichment methods have been developed, including immobilized metal or metal oxide affinity chromatography such as Fe 3+ ion [10] [11], titanium dioxide (TiO 2 ) [12] [13], titanium nanopolymer [14], cation and anion exchange chromatography [15] [16] [17], antibody capture [18] [19] [20], calcium phosphate precipitation, chemical derivation [21] [22], and the combination of these methods [23]. TiO 2 method gains popularity because of high selectivity, recovery, and reproducibility. "
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