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Publications

First Author

Color Schemes

Mango: a bias-correcting ChIA-PET analysis pipeline

Phanstiel, D.H., Boyle,A.P., Heidari, N., Snyder, M.P. Bioinformatics (2015)

Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) is an established method for detecting genome-wide looping interactions at high resolution. Current ChIA-PET analysis software packages either fail to correct for non-specific interactions due to genomic proximity or only address a fraction of the steps required for data processing. We present Mango, a complete ChIA-PET data analysis pipeline that provides statistical confidence estimates for interactions and corrects for major sources of bias including differential peak enrichment and genomic proximity.

Comparison to the existing software packages, ChIA-PET Tool and ChiaSig revealed that Mango interactions exhibit much better agreement with high-resolution Hi-C data. Importantly, Mango executes all steps required for processing ChIA-PET datasets, whereas ChiaSig only completes 20% of the required steps. Application of Mango to multiple available ChIA-PET datasets permitted the independent rediscovery of known trends in chromatin loops including enrichment of CTCF, RAD21, SMC3 and ZNF143 at the anchor regions of interactions and strong bias for convergent CTCF motifs.

Mango is open source and distributed through github at https://github.com/dphansti/mango.

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Color Schemes

Sushi.R: flexible, quantitative and integrative genomic visualizations for publication-quality multi-panel figures

Phanstiel, D.H., Boyle A.P., Araya C.L., Snyder M.P. Bioinformatics (2014)

Interpretation and communication of genomic data require flexible and quantitative tools to analyze and visualize diverse data types, and yet, a comprehensive tool to display all common genomic data types in publication quality figures does not exist to date. To address this shortcoming, we present Sushi.R, an R/Bioconductor package that allows flexible integration of genomic visualizations into highly customizable, publication-ready, multi-panel figures from common genomic data formats including Browser Extensible Data (BED), bedGraph and Browser Extensible Data Paired-End (BEDPE). Sushi.R is open source and made publicly available through GitHub (https://github.com/dphansti/Sushi) and Bioconductor (http://bioconductor.org/packages/release/bioc/html/Sushi.html).

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Color Schemes

Genome-wide map of regulatory interactions in the human genome

Heidari N.*, Phanstiel, D.H.*, He C., Grubert F., Jahanbani F., Kasowski M., Zhang M.Q., Snyder M.P. Genome Research (2014)

Increasing evidence suggests that interactions between regulatory genomic elements play an important role in regulating gene expression. We generated a genome-wide interaction map of regulatory elements in human cells (ENCODE tier 1 cells, K562, GM12878) using Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) experiments targeting six broadly distributed factors. Bound regions covered 80% of DNase I hypersensitive sites including 99.7% of TSS and 98% of enhancers. Correlating this map with ChIP-seq and RNA-seq data sets revealed cohesin, CTCF, and ZNF143 as key components of three-dimensional chromatin structure and revealed how the distal chromatin state affects gene transcription. Comparison of interactions between cell types revealed that enhancer-promoter interactions were highly cell-type-specific. Construction and comparison of distal and proximal regulatory networks revealed stark differences in structure and biological function. Proximal binding events are enriched at genes with housekeeping functions, while distal binding events interact with genes involved in dynamic biological processes including response to stimulus. This study reveals new mechanistic and functional insights into regulatory region organization in the nucleus.

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Color Schemes

Proteomic and phosphoproteomic comparison of human ES and iPS cells

Phanstiel, D.H.*, Brumbaugh J.*, Wenger C.D., Tian S., Probasco M.D., Bailey D.J., Swaney D.L., Tervo M.A., Bolin J.M., Ruotti V., Stewart R., Thomson J.A., Coon J.J. Nature Methods (2011)

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

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Color Schemes

Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells

Phanstiel, D.H., Brumbaugh, J., Berggren, W.T., Conard, K., Feng, X., Levenstein, M.E., McAlister, G.C., Thomson, J.A., Coon, J.J.: Proceedings of the National Academy of Sciences (2008)

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

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Color Schemes

Peptide quantification using 8-plex isobaric tags and electron transfer dissociation tandem mass spectrometry

Phanstiel, D.H., Unwin, R., McAlister, G.C., Coon, J.J. Analytical Chemistry (2009)

Isobaric tags for absolute and relative quantitation (iTRAQ) allow for simultaneous relative quantification of peptides from up to eight different samples. Typically peptides labeled with 8-plex iTRAQ tags are pooled and fragmented using beam-type collision activated dissociation (CAD) which, in addition to cleaving the peptide backbone bonds, cleaves the tag to produce reporter ions. The relative intensities of the reporters are directly proportional to the relative abundances of each peptide in the solution phase. Recently, studies using the 4-plex iTRAQ tagging reagent demonstrated that electron transfer dissociation (ETD) of 4-plex iTRAQ labeled peptides cleaves at the N-C alpha bond in the tag and allows for up to three channels of quantification. In this paper we investigate the ETD fragmentation patterns of peptides labeled with 8-plex iTRAQ tags. We demonstrate that upon ETD, peptides labeled with 8-plex iTRAQ tags fragment to produce unique reporter ions that allow for five channels of quantification. ETD-MS/MS of these labeled peptides also produces a peak at 322 m/z which, upon resonant excitation (CAD), gives rise to all eight iTRAQ reporter ions and allows for eight channels of quantification. Comparison of this method to beam-type CAD quantification shows a good correlation (y = 0.91x + 0.01, R(2) = 0.9383).

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Color Schemes

Peptide and protein quantification using iTRAQ with electron transfer dissociation

Phanstiel, D.H., Zhang, Y., Marto, J.A., Coon, J.J. Journal of The American Society for Mass Spectrometry (2008)

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c- and z-type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-C(alpha) bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error <10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.

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Contributing Author

  • Grubert F., Zaugg J.B., Kasowski M., Ursu O., Spacek D.V., Martin A.R., Greenside P., Srivas R, Phanstiel DH, Pekowska A, Heidari N, Euskirchen G, Huber W, Pritchard JK, Bustamante CD, Steinmetz LM, Kundaje A, Snyder M. Genetic Control of Chromatin States in Humans Involves Local and Distal Chromosomal Interactions. Cell. 2015 Aug 27;162(5):1051-65. doi: 10.1016/j.cell.2015.07.048. Epub 2015 Aug 20. PubMed PMID: 26300125; PubMed Central PMCID: PMC4556133.

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  • Chen, R., Giliani, S., Lanzi, G., Mias, G.I., Lonardi, S., Dobbs, K., Manis, J., Im, H., Gallagher, J.E., Phanstiel, D. H., Euskirchen, G., Lacroute, P., Betinger, K., Moratio, D., Weinacht, K., Montin, D., Gallo, E., Mangili, G., Porta, F., Notarangelo, L.D., Pedreti, S., Al-Herz, W., Alfahdli, W., Comeau, A.M., Traister, R.S., Pai, S.Y., Carella, G., Faccheti, F., Nadeau, K.C., Snyder, M., Notarangelo, L.D.: Whole-exome sequencing identifies tetratricopeptide repeat domain 7A (TTC7A) mutations for combined immunodeficiency with intestinal atresias. J. Allergy Clin. Immunol. 132(3), 656–664 (2013). [PubMed Central:PMC3759618] [DOI:10.1016/j.jaci.2013.06.013] [PubMed:23830146]

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  • Brumbaugh, J., Rose, C.M., Phanstiel, D. H., Thomson, J.A., Coon, J.J.: Proteomics and pluripotency. Crit. Rev. Biochem. Mol. Biol. 46(6), 493–506 (2011). [PubMed Central:PMC3223296] [DOI:10.3109/10409238.2011.624491] [PubMed:21999516]

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  • Wenger, C.D., Lee, M.V., Hebert, A.S., McAlister, G.C., Phanstiel, D. H., Westphall, M.S., Coon, J.J.: Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat. Methods 8(11), 933–935 (2011). [PubMed Central:PMC3205195] [DOI:10.1038/nmeth.1716] [PubMed:21963608]

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  • McAlister, G.C., Phanstiel, D. H., Brumbaugh, J., Westphall, M.S., Coon, J.J.: Higher-energy collision activated dissociation without a dedicated collision cell. Mol. Cell Proteomics 10(5), 111–009456 (2011). [PubMed Central:PMC3098599] [DOI:10.1074/mcp.O111.009456] [PubMed:21393638]

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  • Wenger, C.D., Phanstiel, D. H., Lee, M.V., Bailey, D.J., Coon, J.J.: COMPASS: a suite of pre- and postsearch proteomics software tools for OMSSA. Proteomics 11(6), 1064–1074 (2011). [PubMed Central: PMC3049964] [DOI:10.1002/pmic.201000616] [PubMed:21298793]

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  • McAlister, G.C., Phanstiel, D. H., Wenger, C.D., Lee, M.V., Coon, J.J.: Analysis of tandem mass spectra by FTMS for improved large-scale proteomics with superior protein quantification. Anal. Chem. 82(1), 316–322 (2010). [PubMed entral:PMC2800853] [DOI:10.1021/ac902005s] [PubMed:19938823]

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  • Brumbaugh, J., Phanstiel, D. H., Coon, J.J.: Unraveling the histone’s potential: a proteomics perspective. Epigenetics 3(5), 254–257 (2008). [PubMed Central:PMC2662511] [PubMed:18849650]

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  • McAlister, G.C., Berggren, W.T., Griep-Raming, J., Horning, S., Makarov, A., Phanstiel, D. H., Stafford, G., Swaney, D.L., Syka, J.E., Zabrouskov, V., Coon, J.J.: A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer. J. Proteome Res. 7(8), 3127–3136 (2008). [PubMed Central:PMC2601597] [DOI:10.1021/pr800264t][PubMed:18613715]

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  • Faca, V.M., Song, K.S., Wang, H., Zhang, Q., Krasnoselsky, A.L., Newcomb, L.F., Plentz, R.R., Gurumurthy, S., Redston, M.S., Pitieri, S.J., Pereira-Faca, S.R., Ireton, R.C., Katayama, H., Glukhova, V., Phanstiel, D. H., Brenner, D.E., Anderson, M.A., Misek, D., Scholler, N., Urban, N.D., Barneti, M.J., Edelstein, C., Goodman, G.E., Thornquist, M.D., McIntosh, M.W., DePinho, R.A., Bardeesy, N., Hanash, S.M.: A mouse to human search for plasma proteome changes associated with pancreatic tumor development. PLoS Med. 5(6), 123 (2008). [PubMed Central:PMC2504036][DOI:10.1371/journal.pmed.0050123] [PubMed:18547137]

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  • Pereira-Faca, S.R., Kuick, R., Puravs, E., Zhang, Q., Krasnoselsky, A.L., Phanstiel, D. H., Qiu, J., Misek, D.E., Hinderer, R., Tammemagi, M., Landi, M.T., Caporaso, N., Pfeiffer, R., Edelstein, C., Goodman, G., Barneti, M., Thornquist, M., Brenner, D., Hanash, S.M.: Identification of 14-3-3 theta as an antigen that induces a humoral response in lung cancer. Cancer Res. 67(24), 12000–12006 (2007). [DOI:10.1158/0008-5472.CAN-07-2913] [PubMed:18089831]

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  • Faca, V., Pitieri, S.J., Newcomb, L., Glukhova, V., Phanstiel, D. H., Krasnoselsky, A., Zhang, Q., Struthers, J., Wang, H., Eng, J., Fitzgibbon, M., McIntosh, M., Hanash, S.: Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J. Proteome Res. 6(9), 3558–3565 (2007). [DOI:10.1021/pr070233q] [PubMed:17696519]

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  • McAlister, G.C., Phanstiel, D. H., Good, D.M., Berggren, W.T., Coon, J.J.: Implementation of electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer. Anal.Chem. 79(10), 3525–3534 (2007). [PubMed Central:PMC2662514] [DOI:10.1021/ac070020k] [PubMed:17441688]

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  • Faca, V., Coram, M., Phanstiel, D. H., Glukhova, V., Zhang, Q., Fitzgibbon, M., McIntosh, M., Hanash, S.: Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS. J. Proteome Res. 5(8), 2009–2018 (2006). [DOI:10.1021/pr060102+] [PubMed:16889424]

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US Patents

  • Coon, J.J., Phanstiel, Douglas H, Wenger, C.D.: Mass Spectrometry Data Acquisition Mode for Obtaining More Reliable Protein Quantitation. 8455818 B2, April 2013

  • Coon, J., Phanstiel, Douglas H, McAlister, G.: Probability-based Mass Spectrometry Data Acquisition. 8530831 B1, October 2013

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