Panorama: A Targeted Proteomics Knowledge Base

Sharma, Vagisha; Eckels, Josh; Taylor, Greg; Shulman, Nicholas; Stergachis, Andrew B.; Joyner, Shannon; Yan, Ping; Whiteaker, Jeffrey R.; Halusa, Goran N.; Schilling, Birgit; Gibson, Bradford W.; Colangelo, Christopher M.; Paulovich, Amanda G.; Carr, Steven A.; Jaffe, Jacob D.; MacCoss, Michael J.; MacLean, Brendan

doi:10.1021/pr5006636

Cited by 208 publications

(192 citation statements)

References 20 publications

(29 reference statements)

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“…The Skyline documents containing the targeted mass spectrometry data (including quantification of HER2 peptides in the cohort samples, linearity and LLOQ data) have been deposited on the Panorama server (25). Data can be accessed on the Panorama Public website using the following link: https://panoramaweb.org/labkey/Steiner.url.…”

Section: Methodsmentioning

confidence: 99%

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Steiner

Tille

Lamerz

et al. 2015

Molecular & Cellular Proteomics

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The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selected reaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R 2 : 0.99 -1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website. MS based proteomics has traditionally been used to investigate complex biological systems, such as cell lines, plasma, or fresh-frozen tissues (1, 2). In the last decade however, MS proteomics has extended to the analysis of formalin-fixed paraffin-embedded (FFPE) 1 tissues (3). Formalin fixation is the gold standard for sample storage in clinical pathology because it allows optimal preservation of the morphological features of the tissue and it is economically attractive (storage at room temperature over several years or decades) (4). Sev-

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Section: Methodsmentioning

confidence: 99%

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Steiner

Tille

Lamerz

et al. 2015

Molecular & Cellular Proteomics

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“…The 55-min gradient started with 8% B, which increased to 26% in 35 min and then to 43% in 15 min. The SRM assays for Synechocystis proteins were loaded from the Panorama Public (Sharma et al, 2014) data repository, and the assays for the missing targets were developed according to the published protocol. Data analysis was performed in RStudio with MSstats algorithm version 3.5.1 (Choi et al, 2014).…”

Section: Targeted Quantificationmentioning

confidence: 99%

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

et al. 2017

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H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a wellcharacterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (DndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the DndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-1 2 complex, and decreases the expression of several transporters in ambient CO 2 growth conditions.

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“…Cells then were dislodged, pipetted into a 1.5-mL MCF tube, and centrifuged for 5 s at 3,540 × g. Dishes were tilted, excess PBS was pipetted off, 200 μL of 10% (vol/vol) trichloroacetic acid/90% (vol/vol) acetone/0.2% DTT was added, and samples were incubated at −20°C for 1 h. Protein extraction, protein assay, and insolution trypsin digestion were performed as previously reported (114). The spectral library used for Skyline analysis of SWATH-MS data (115) and results are available in Panorama public (116) at https://panoramaweb.org/labkey/ XW2016-1.url.…”

Section: Methodsmentioning

confidence: 99%

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Wang

Kültz

2017

Proc. Natl. Acad. Sci. U.S.A.

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Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of O. mossambicus. Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1. Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation). biochemical evolution | compatible osmolytes | osmotic stress signaling | enhancer | CRE

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Panorama: A Targeted Proteomics Knowledge Base

Cited by 208 publications

References 20 publications

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

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