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PTMScan Direct: Identification and Quantification of Peptides from Critical Signaling Proteins by Immunoaffinity Enrichment Coupled with LC-MS/MS*

Open AccessPublished:February 09, 2012DOI:https://doi.org/10.1074/mcp.M111.015883
      Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
      The development of efficacious compounds to fight diseases including cancer, developmental defects, neurodegenerative disease, infectious disease, and metabolic disorders is an area of intense focus in both academic and industrial laboratories. An understanding of the cellular signaling pathways underlying these various disease states is critical to effective drug development programs, both in predicting response to compounds and in anticipating off target effects. Post-translational modification of signaling proteins involved in these pathways is a critical factor in determination of activity, localization, and protein-protein interactions in disease as well as other experimental systems such as protein overexpression, knockdown, or studies of the effects of tissue microenvironment.
      Decades of work have provided insight into some of the mechanisms underlying various disease states, such as the dependence on tyrosine kinase activity for growth and survival of some cancer types (
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      In the past, the study of protein activity in complex diseases and cellular signaling pathways has either focused on a few proteins known to be critical to the system being studied or has employed proteomic methods that provide rich data sets that randomly sample the proteome. The detailed study of one or a few specific proteins has the advantage of focusing on known pathway components but suffers from an inability to sample many data points from complex systems. Previous proteomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS)
      The abbreviations used are:
      LC-MS/MS
      liquid chromatography tandem mass spectrometry
      EGFR
      epidermal growth factor receptor
      PDGFR
      platelet-derived growth factor receptor
      PI3K
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      RTK
      receptor tyrosine kinase
      MAP
      mitogen-activated protein
      mTOR
      mammalian target of rapamycin.
      1The abbreviations used are:LC-MS/MS
      liquid chromatography tandem mass spectrometry
      EGFR
      epidermal growth factor receptor
      PDGFR
      platelet-derived growth factor receptor
      PI3K
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      RTK
      receptor tyrosine kinase
      MAP
      mitogen-activated protein
      mTOR
      mammalian target of rapamycin.
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      Assessing the activity of cediranib, a VEGFR-2/3 tyrosine kinase inhibitor, against VEGFR-1 and members of the structurally related PDGFR family.
      ). Current methods allow for simultaneous identification and quantification of thousands of post-translationally modified peptides across serine, threonine, and tyrosine phosphorylation, as well as ubiquitination, neddylation, ISGylation (ISG15 modification), and acetylation (www.cellsignal.com/services). A limitation of these strategies is that the list of peptides to be quantified is variable (data-dependent) and based upon factors such as the cell line or tissue type profiled, treatment conditions, motif antibody employed, and duty cycle limitations of the mass spectrometer. Development of methods that focus on a defined set of peptides from important signaling proteins would bypass this limitation and provide complimentary data to traditional data-dependent proteomic analysis.
      To address this need, a novel antibody-based method, called PTMScan Direct, has been developed for identification and quantitation of post-translationally modified peptides. This method, rather than targeting specific peptide sequence motifs, targets peptides derived from proteins that are critical signaling nodes of various pathways or derived from a single protein type, such as kinases. Four PTMScan Direct reagents have been generated to date, including PTMScan Direct: Multipathway, Ser/Thr Kinase, Akt/PI3K Pathway, and Tyr Kinase reagents. The Multipathway reagent detects core proteins of many different cell signaling pathways, such as Akt signaling, MAP kinase signaling, cell cycle regulation, apoptosis, and transforming growth factor-β signaling, among others. The Ser/Thr Kinase reagent profiles over 300 phosphorylation sites on 130 serine and threonine kinases. The Tyr Kinase reagent detects over 600 tyrosine phosphorylation sites on 120 tyrosine kinases and PI3Ks. The Akt/PI3K Pathway reagent detects 296 phosphorylation sites on 105 proteins involved in Akt and PI3K-dependent signaling (more detailed information for each reagent is available at http://www.cellsignal.com/services/direct_overview.html).
      The proteins and corresponding sites detected by each reagent have been determined by a validation strategy that includes profiling human cancer cell lines and mouse tissues. The utility of PTMScan Direct is demonstrated with an investigation of signaling changes in human cancer cell lines in response to receptor tyrosine kinase inhibition using the Multipathway reagent, as well as profiling of kinase phosphorylation in mouse tissues using the Ser/Thr Kinase reagent. Together, this work demonstrates that PTMScan Direct is a powerful method that combines the high number of data points assayed and sensitivity of LC-MS/MS analysis with the specificity of antibody-based methods, allowing quantitative profiling of hundreds of data points that is focused on the signaling pathway or pathways of interest.

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