Computer aided manual validation of mass spectrometry-based proteomic data

Curran, Timothy G.; Bryson, Bryan; Reigelhaupt, Michael; Johnson, Hannah; White, Forest M.

doi:10.1016/j.ymeth.2013.03.004

Cited by 29 publications

(25 citation statements)

References 17 publications

(16 reference statements)

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“…Raw mass spectral data files were searched against SwissProt database containing Homo sapiens protein sequences using Mascot version 2.4. MS/MS spectra of phosphorylated peptides observed in multiple biological replicates were validated using computer-assisted manual validation (CAMV) software to confirm phosphorylation assignment and isolation purity (30). From accepted scans, reporter ion quantification was extracted, isotope corrected, and normalized based on median relative protein quantification ratios.…”

Section: Methodsmentioning

confidence: 99%

Early signaling dynamics of the epidermal growth factor receptor

Reddy

Gajadhar

Swenson

et al. 2016

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

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Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.signal transduction | tyrosine phosphorylation | epidermal growth factor receptor | mass spectrometry T he EGF receptor (EGFR) sits atop a complex signaling network that controls cell behavior in response to environmental cues. Cascades of posttranslational modifications initiated from EGFR, notably phosphorylation on tyrosine, serine, and threonine, influence protein-protein interactions and enzymatic activity to activate transcriptional programs that regulate proliferation, differentiation, and apoptosis (1). Mutation or overexpression of EGFR has been identified as an oncogenic driver for many tumor types, making it an attractive target for anticancer therapies (2).Building on decades of specific characterization using traditional biochemistry techniques, platforms such as protein microarrays and mass spectrometry have allowed systems biology to provide a comprehensive picture of intact and aberrant network behavior, which can be used to establish design criteria for therapeutic interventions (3). Manipulating components within the network experimentally and computationally has uncovered many features of the system that influence behaviors such as proliferation and survival (4-6). With many phenotypic responses occurring on the order of hours to days, most phosphorylation measurements have been on the timescale of minutes to...

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Section: Methodsmentioning

confidence: 99%

Early signaling dynamics of the epidermal growth factor receptor

Reddy

Gajadhar

Swenson

et al. 2016

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

Self Cite

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“…22 Curated, annotated spectra of the 402 identified and quantified phosphotyrosine peptides can be found in Supporting Information Figure S1. Unsupervised hierarchical clustering of the mean normalized and log 2 -transformed phosphotyrosine and protein expression quantitative iTRAQ data was generated using GENE-E ().…”

Section: Methodsmentioning

confidence: 99%

Quantitative Analysis of Signaling Networks across Differentially Embedded Tumors Highlights Interpatient Heterogeneity in Human Glioblastoma

Johnson

White

2014

J. Proteome Res.

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Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs immediate flash freezing (iFF) in LN2 on protein expression and phosphorylation-mediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large interpatient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen.

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“…Peptide sequences were initially filtered for phosphorylated sequences above a MASCOT ion score cutoff of 20, which were detected in each of two process replicates. The spectra of this initial peptide substrate list were evaluated for accurate database identification with a computer-assisted manual validation software (Curran et al 2013). MS/MS spectra of the phosphorylated peptides were manually inspected to confirm correct peptide identification and phosphorylation site localization.…”

Section: Labeling and Mass Spectrometry Identification Of Ime2 Substrmentioning

confidence: 99%

A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern

et al. 2013

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Production of haploid gametes from diploid progenitor cells is mediated by a specialized cell division, meiosis, where two divisions, meiosis I and II, follow a single S phase. Errors in progression from meiosis I to meiosis II lead to aneuploid and polyploid gametes, but the regulatory mechanisms controlling this transition are poorly understood. Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I-meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 59 untranslated region (UTR) of CLB3. At the onset of meiosis II, Ime2 kinase activity rises and triggers a decrease in Rim4 protein levels, thereby alleviating translational repression. Our results elucidate a novel developmentally regulated translational control pathway that establishes the meiotic chromosome segregation pattern.

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Computer aided manual validation of mass spectrometry-based proteomic data

Cited by 29 publications

References 17 publications

Early signaling dynamics of the epidermal growth factor receptor

Early signaling dynamics of the epidermal growth factor receptor

Quantitative Analysis of Signaling Networks across Differentially Embedded Tumors Highlights Interpatient Heterogeneity in Human Glioblastoma

A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern

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