CPTAC Assay Portal: a repository of targeted proteomic assays

Whiteaker, Jeffrey R.; Halusa, Goran N.; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A.; Kennedy, Jacob J.; Mani, D. R.; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Paulovich, Amanda G.; Abbatiello, Susan E.; Boja, Emily S.; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri R.; Ellis, Matthew J.; Fenyö, David; Hiltke, Tara; Ketchum, Karen A.; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C.; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael J.; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly V.; Scott, Mitchell G.; Smith, Richard; Thomas, Stefani N.; Townsend, R. Reid; Whiteley, Gordon; Wu, Chen; Zhang, Hui; Zhang, Zhen

doi:10.1038/nmeth.3002

Cited by 159 publications

(169 citation statements)

References 13 publications

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“…Finally, MRM-based assays are highly distributable among laboratories (22,25), and the assays validated in this study have been made publicly available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay Portal (http://assays.cancer.gov) (42), where SOPs, reagents, and assay characterization data for these assays can be viewed and downloaded. The downloadable assay information enables any research laboratory to prepare samples, which can be transferred to core facilities for MRM analysis, as is currently done for most genomic analyses.…”

Section: Discussionmentioning

confidence: 99%

“…Assays that failed validation because of high day-to-day variability were generally associated with endogenous signals at or near the limit of detection or had relatively high hydrophobicity, likely resulting in instability of the peptide standards because of adsorption losses or solubility issues. Characterization data and standard operating protocols for all validated IMAC-MRM assays have been made available as a resource for the community via the CPTAC assay portal (assays.cancer.gov) (42), where in addition to viewing the data, documentation describing the characterization experiments can be browsed and downloaded.…”

Section: Figmentioning

confidence: 99%

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Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Kennedy

Yan

Zhao

et al. 2016

Molecular & Cellular Proteomics

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A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatographymultiple reaction monitoring assay targeting phosphoanalytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of >3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intraassay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography-multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in "systems biology" approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1, 2), as well as poor performance of affinity reagents used in traditional proteomic assay and detection platforms (3, 4). There is an imminent need for high quality assays, including highly characterized standards and detailed documentation of processes and procedures (5). To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation.Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto "gold standard," it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multipl...

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Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Kennedy

Yan

Zhao

et al. 2016

Molecular & Cellular Proteomics

Self Cite

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“…Overall, the criteria of importance are ultimately governed by the purpose of the intended assay, with more strict criteria generally required for Tier 1 assays than for Tier 3. Additional workflow improvements (e.g., automated sample handling and processing with robotics, such as the AssayMap Bravo [71] or the Tecan Freedom Evo [107]) have helped streamline development and application, while public repositories of validated MS/MS assays have been created to aid standardization [108][109][110].…”

Section: Expert Commentarymentioning

confidence: 99%

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

Percy¹,

Byrns

Pennington

et al. 2016

Expert Review of Proteomics

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This article reviews the status, challenges, requirements, and potential of translating current, MS-based methods to the clinical laboratory. The described methods are discussed and contrasted within a fit-for-purpose approach, while different resources for quality control, quantitative analysis, and data interpretation are additionally provided. Expert commentary: Although great strides have been made over the past five years in developing reliable quantitative assays for plasma protein biomarkers, it is crucial for investigators to have an understanding of the clinical validation process, a major roadblock in translational research. Continued progress in method design and validation of protein assays is necessary to ultimately achieve widespread adoption and regulatory approval.

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“…For example, fluctuations in protein expression during different disease states or in response to biological intervention can be detected. Advances in the latest technologies, software platforms and online repository databases have not only increased the capabilities of this field but also made it more accessible to clinical researchers [1][2][3]. Indeed, applying a proteomic approach to cardiovascular medicine has proven to be very successful.…”

Section: Introductionmentioning

confidence: 99%

Psoriatic Arthritis Under a Proteomic Spotlight: Application of Novel Technologies to Advance Diagnosis and Management

et al. 2015

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Psoriatic arthritis is a form of inflammatory arthritis that is frequently associated with psoriasis. Individuals with this disease present with heterogeneous clinical manifestations, making it challenging to diagnose and select optimal treatment strategies. Perhaps, not unsurprisingly, there are currently no molecular diagnostic or prognostic tests to confirm if a patient has the disease or predict how they may respond to therapy. Instead, a range of classification criteria have been developed, and the experience of the treating clinician is heavily relied upon. It is therefore widely accepted that there is a significant and as yet unmet need for effective molecular markers in psoriatic arthritis. Protein mediators drive disease pathogenesis and, therefore, represent logical potential biomarkers. Indeed, significant advances have recently been made by the introduction of multiplexed protein biomarker tests for monitoring disease activity in rheumatoid arthritis. At the same time, recent advances in proteomics have enhanced the capabilities for the detection and discovery of protein biomarkers. These advances offer renewed opportunities for the development of multi-protein biomarker signatures to support clinical decision-making in the diagnosis, prognosis and treatment of psoriatic arthritis. This review summarises the pathogenesis of psoriatic arthritis, highlighting specific areas of unmet clinical need. Furthermore, it seeks to illustrate how the latest developments in proteomic technologies could be used to enhance our understanding of the molecular pathology of psoriatic arthritis and improve clinical outcomes and quality of life for patients.

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CPTAC Assay Portal: a repository of targeted proteomic assays

Cited by 159 publications

References 13 publications

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

Psoriatic Arthritis Under a Proteomic Spotlight: Application of Novel Technologies to Advance Diagnosis and Management

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