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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Available from: Albrecht Moritz, Sep 30, 2015
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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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    ABSTRACT: Aberrant expression and activation of FGFR3 is associated with disease states including bone dysplasia and malignancies of bladder, cervix, and bone marrow. MS analysis of protein-phosphotyrosine in multiple myeloma cells revealed a prevalent phosphorylated motif, D/EYYR/K, derived from the kinase domain activation loops of tyrosine kinases including FGFR3 corresponding to a recognition sequence of phosphotyrosine phosphatases PTPN1. Knockdown of PTPN1 or the related enzyme PTPN2 by RNAi resulted in ligand-independent activation of FGFR3. Modulation of FGFR3 activation loop phosphorylation by both PTPN1 and PTPN2 was a function of receptor trafficking and PTP compartmentalization. The FGFR3 activation loop motif DYYKK650 is altered to DYYKE650 in the oncogenic variant FGFR3K650E, and consequently it is constitutively fully activated and unaffected by activation loop phosphorylation. FGFR3K650E was nevertheless remarkably sensitive to negative regulation by PTPN1 and PTPN2. This suggests that in addition to modulating FGFR3 phosphorylation, PTPN1 and PTPN2 constrain the kinase domain by fostering an inactive-state. Loss of this constraint in response to ligand or impaired PTPN1/N2 may initiate FGFR3 activation. These results suggest a model wherein PTP expression levels may define conditions that select for ectopic FGFR3 expression and activation during tumorigenesis.This article is protected by copyright. All rights reserved
    Proteomics 01/2015; 15(2-3). DOI:10.1002/pmic.201400259 · 3.81 Impact Factor
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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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    ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a non-phosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 non-redundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain.This article is protected by copyright. All rights reserved
    Proteomics 10/2014; 15(2-3). DOI:10.1002/pmic.201400171 · 3.81 Impact Factor
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    • "Cells were washed and suspended in PBS and treated with 100 ng/ml EGF for 5 or 15 min. For tyrosine inhibition of phosphatases , cells were treated with 1 mM pervanadate and 50 ng/ml calyculin A for 15 min at 37 C as described earlier (Rush et al., 2005). "
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    ABSTRACT: Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.
    Cell Reports 08/2014; 8(5). DOI:10.1016/j.celrep.2014.07.036 · 8.36 Impact Factor
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