Harsha Gunawardena

harshapg@excite.com

harshapg@med.unc.edu

 

In the past 3 years I have been involved in a wide variety of proteomics and protein characterization projects at UNC and as a part of multi-site collaborations (i.e., ENCODE, CPTAC, Thermo BRIMS). I was involved in setting-up two laboratories and coordinating its day-to-day operations. The labs are equipped with high-end mass spectrometers such as Oribtrap XL,Orbitrap Velos, Q Exactive, TSQ-triple quads. The efficient processing of large volumes of proteomic data is made possible by several commercial protein identification and quantification programs that use both database search and denovo sequencing algorithms. We are performing systems biology and statistics using suite software to produce custom reports containing information about the biological system in study.

 

As a part of the ENCODE (Encyclopedia of DNA elements) consortium we have conducted in-depth proteomic and proteogenomic analysis of the tier 1-3 ENCODE cell-lines. Proteomics has played a major role in the ENCODE projects resulting in the identification of close to 10,000 protein groups per cell-line. We have also catalogued proteomic changes at both the whole-cell level and at sub-cellular resolution. A variety of sample pre-fractionation methods were utilized i.e., SCX, SDS-PAGE, GELFrEE-FASP  prior to LC-MS analysis on Oribtrap-based mass spectrometer. The mass spectra are analyzed using a suite of search engines i.e., Mascot, Andromeda, Sequest, X!Tandem, against annotated protein sequence databases and annotated transcripts. In collaboration, these mass-spectra have also been subjected to whole-genome scale proteogenomic mapping via a 6-frame translation strategy. Through a machine learning HMM algorithms and more advanced IMP algorithms, proteomics scale data are mapped to unannotated regions of the genome. This technology has enabled the verification of pseudo genes, splice variants, and protein level evidence of inter-genic regions. The information resulting from this analysis is annotated as a MS-BED track in the UCSB genome browser.  The mass spectrometry data is a resource for biologists with multi-OMIC’s’ data. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

We are also one of the five main proteome characterization centers for the CPTAC (clinical proteomic tumor analysis consortium). Our initial label-free quantitative workflows applied to mouse tissue proteomes of breast cancer xenograft has resulted in close-to 10 000 quantifiable proteins. We have undertaken studies to ascertain protein stability, tumor micro heterogeneity, and deep-sequencing of reference standard material derived from basal and luminal breast cancer sub-types. We are also exploring the figures of merit of label-free quantitation platforms, super SILAC and iTRAQ applied to tissue, cell-lines, and IP-MS in the context of breast cancer tissue proteomics.

 

Publications

 

·         Landsacpe of Transcription in Human Cells (2012) Nature  Accepted

 

·         The Encode project consortium “Initial Analysis of the Encyclopedia of DNA Elements in the Human Genome” (2012) Nature Accepted

 

·         Balazs Banfai*, Hui Jia*, Jainab Khatun*, Emily Wood, Christopher Maier, Will Gundling, Anshul Kundaje, Peter Bickel, Harsha P. Gunawardena, Yanbao Yu, Ling Xie, Krysztof , Krajewski, Brian Strahl, Xian Chen, Morgan Giddings, Ben Brown, Leonard Lipovich “Long non-coding RNAs are rarely translated in two human cell lines (GRCP025) 2012 Genome Research Accepted

 

·         Jainab Khatun1, Yanbao Yu2,  John Wrobel1, Christopher Maier2, Brian Risk1,  Harsha Gunawardena2,  Ashley Secrest1, Wendy Spitzer1, Dennis Crenshaw1, Xian Chen2 and Morgan C. Giddings1,2* “Whole human genome proteogenomic mapping: identifying protein-coding regions” 2012 In Genome Biology In revision

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

·         Harsha P. Gunawardena, Min He, Paul A. Chrisman, Sharon J. Pitteri, Jason M. Hogan, Brittany D. M. Hodges, and Scott A. McLuckey, J. Am. Chem. Soc. 2005, 127, 12627-12639 “Electron Transfer versus Proton Transfer in Gas-Phase Ion-Ion Reactions of Polyprotonated Peptides”

 

 

 

 

 

Conference Proceedings

 

 

 

·         Barbara Frewen1; Scott Peterman2; Eugene Ciccimaro2; Parag Mallick3; Sebastien Gallien4; Bruno Domon4; Mohit Jain8; Tong Lin8; Hong Li8; Brian Hood6; Tom Conrads6; Chris Smith7; Ihor Batruch7; Andrei Drabovich7; Vathany Kulasingam7; Harsha Gunawardena5; Xian Chen5; Vincent Chen9; Leonard Foster9; David Sarracino1; Bryan Krastins1; Eleftherios Diamandis7; Michael Athanas1; Cynthia Lorang3; Maryann Vogelsang1; Alejandra Garces1; Mary Lopez1; Amol Prakash1 Improved Spectral Libraries with Data Quality Requirements, Internal Standards and Normalized Results World  HUPO, Boston,  CA (2012).

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·         Yanbao Yu ; Harsha P. Gunawardena ; Xian Chen “Improving Peptide Identification Hit-Rate via Accounting for Chemical Artifacts in Global Proteomics” American Society for Mass Spectrometry, Vancouver, Canada (2012).

 

·         Barbara Frewen1; Scott Peterman2; Eugene F. Ciccimaro2; Parag Mallick3; Sebastien Gallien4; Bruno Domon4; Mohit R Jain7; Tong Lin7; Hong Li7; Chris Smith6; Ihor Batruch6; Andrei Drabovich6; Vathany Kulasingam6; Eleftherios Diamandis6; Harsha Gunawardena 5; Xian Chen5; Cynthia Lorang3; Leonard Foster8; Vincent Chen8; Maryann Vogelsang1; Alejandra Garces1; David Sarracino 1; Michael Athanas1; Bryan Krastins1; Mary F Lopez1; Amol Prakash1 “Improved Spectral Libraries with Data Quality Requirements, Internal Standards and Normalized Results” American Society for Mass Spectrometry, Vancouver, Canada (2012).

 

·         Harsha P. Gunawardena 1; David Fenyo3; Sherri Davis2; Elaine Mardis2; Jeanne Rumsey2; Petra Erdmann-Gilmore2; Jainab Khatun4; John Wrobel4; Brian Risk4; Morgan Giddings4; Matthew J. Ellis2; R. Reid Townsend2; Xian Chen1 “Evaluating Multiple Label-Free Quantitative Proteomic Platforms: A case study of Basal versus Luminal Human Breast Cancer Tumor Xenografts in Mouse”, American Society for Mass Spectrometry, Vancouver, Canada (2012).

 

·         Morgan Giddings1; Reid Townsend2; Matthew Ellis2; Xian Chen3; John Wrobel1; Brian Risk1; Jainab Khatun1; Sherri Davies2; Elaine Mardis2; Jeanne Rumsey Petra Erdmann-Gilmore2; Yanbao Yu3; Harsha Gunawardena3 “Pseudogenes, alternative splices, and SNPs found in breast tumors via “personal genome” proteogenomic mapping” US HUPO, San Fransisco,  CA (2012).

 

·         Cristina Osorio1,2, Paola Zapata1,5, Harsha P. Gunawardena2,4, Mihaela Mocanu1,2, Carolina Londono1, and Oscar Alzate1,2,3* “The proteome of the Exosporium and the Parasporal Crystals of Bacillus thuringiensis BGSC-4AW1 (var. Andalousiensis) A cancer cell-killing Cry protein”

 

·         Barbara Frewen1,8, Scott Peterman2, Gene Ciccimaro2, Parag Mallick3, Sebastien Gallien4, Bruno Domon4, Harsha Gunawardena5, Xian Chen5,Chris Smith6, Ihor Batruch6, Andrei Drabovich6, Vathany Kulasingam6,  Eleftherios P. Diamandis6, Leonard Foster7, Michael Athanas8, Bryan Krastins8, Alejandra Garces8, David Sarracino8, Mary Lopez8, Amol Prakash8 “A sparkplug for clinical assays: progressing towards autonomous global spectral repositories”, MSACL, San Diego, CA, 2012

 

·         Yanbao Yu, Ling Xie, Harsha P. Gunawardena, Christorpher Miers, Jainab Khatun, Maarten Lerkes, , Xian Chen, Morgan Giddings “Microwave-assisted GELFrEE-FASP: A novel integrated platform for large-scale comprehensive proteomics” American Society for Mass Spectrometry, Denver, CO (2011).

 

·         Harsha P. Gunawardena, Yanbao Yu, Xian Chen “Quantitative Proteomic Dissection of the Dynamic Myddasome” American Society for Mass Spectrometry, Denver, CO (2011).

 

·         Harsha P. Gunawardena, Yi Huang, Roma Kenjale, Haiyang Wang, Ling Xie, Xian Chen “LPS-induced Phosphorylation of LRRFIP2 is critical for MyD88-TLR4 Signaling” US HUPO, Raleigh,  NC (2011).

 

·         Christopher Maier, Jainab Khatun, Brian Risk, Yanbao Yu, Harsha Gunawardena, Dennis Crenshaw, John Wrobel, Xian Chen, Morgan Giddings “Uncovering the Hidden Life of the Human Genome by Proteomic Analysis” US HUPO, Raleigh,  NC (2011).

 

·         Harsha P. Gunawardena, Yi Huang, Li Wang, Xian Chen “Label-free quantitation to dissect site-specific phosphorylation stoichiometry and dynamics of signaling proteins involved in inflammation and pathogenesis” American Society for Mass Spectrometry, Salt Lake City, UT (2010).

 

·         Harsha P. Gunawardena, Li Wang, Cui Liu, Ying Du, Xian Chen “Systems investigation of MyD88 dependent phosphoproteome” American Society for Mass Spectrometry, Salt Lake City, UT (2010)

 

·         Heather Kuiper, Harsha P. Gunawardena, Cui Liu, Xian Chen “Quantitative Proteomic Dissection of Epigenetic Changes Associated with LPS-induced Inflammation” American Society for Mass Spectrometry, Salt Lake City, UT (2010)

 

·         Harsha P. Gunawardena, Yanbao Yu, Carol E. Parker, Brain Button, Marian Mullebach, Richard. C. Boucher, Xian Chen “Multiplex Mass Spectrometry Approaches for Analysis of Differential Expression of Proteins in Plasma of Cystic Fibrosis Patients Treated for Lung Disease”. American Chemical Society, Washington DC,  ( 2009)

 

·         Harsha P. Gunawardena, Ling Xie, Yanbao Yu, Heather O’Donohue, Paul B. Manis, Xian Chen “Multiplex iTRAQ-based approach for cochlear nucleus proteomics in a mosue model of age-related hearing loss” American Society for Mass Spectrometry, Philadelphia, PA (2009) 

 

·         Yanbao Yu, Harsha P. Gunawardena*, Xian Chen “A recovery strategy of co-depleted proteins in affinity-based separation workflows for plasma and serum” American Society for Mass Spectrometry, Philadelphia, PA (2009)  [Presenting Author *]

 

·         Harsha P. Gunawardena “Gas-Phase Ion/Ion Chemistry and Mass Spectrometry for the Determination of  Alpha-1 Proteinase Inhibitor Oxidation Sites”  American Society for Mass Spectrometry, Denver, CO (2008) 

 

·         Harsha  P. Gunawardena, Christina Slone “Detecting Protein Carbonylation via Capillary Zone Electrophoresis (CZE)” American Chemical Society, New Orleans, LA (2008)

 

·         Harsha P. Gunawardena  “Investigation of Protein Oxidation by Fixed Charge Derivatization and Capillary Electrophoresis” American Chemical Society, Boston, MA (2007)

 

·         Harsha P. Gunawardena “Gas-Phase Ion Chemistry and Tandem Mass Spectrometry for Determination of  Alpha anti Trypsin Inhibitor Oxidation Sites”  American Chemical Society, Boston, MA (2007)

 

·         Bruce H. Bailey, Mamoun M. Bader, Daniel S. Jones, Harsha P. Gunawardena “Analysis of 1-diphenylamino-4-tricyanoetheneylbenzene by X-ray diffraction” American Chemical Society, Boston, MA (2007)

 

·         Harsha. P. Gunawardena, Judy Fredlin, Koen van der Drift, Kevin Wee “Screening of Disulfide Induced Protein Refolding Intermediates by Capillary Isoelectric Focusing” American Chemical Society, Chicago, IL (2007)

 

·         Jeremiah J. Bowers, Jian Liu, Harsha P. Gunawardena and Scott A. McLuckey “Protein Identification via Ion Trap Collision-induced Dissociation and Examination of Low Mass Product Ion Spectra  54th American Society for Mass Spectrometry Conference, Seattle, WA (2006)

 

·         Hongling Han, Xiarong Liang, Harsha P. Gunawardena, Yu Xia, Scott A. McLuckey “Electron Transfer Ion/Ion reactions of Doubly and Singly Charged Peptides: Effect of Cooling Gas Pressure” 54th American Society for Mass Spectrometry Conference, Seattle, WA (2006)

 

·         Harsha P. Gunawardena, and Scott A. McLuckey “Electron Transfer Dissociation of Disulfide Linked Polypeptides: Effect of Charge State and Charge Site  54th American Society for Mass Spectrometry Conference, Seattle, WA (2006)

 

·         Tegafaw. T. Mekecha, Harsha P. Gunawardena, and Scott A. McLuckey “Altering Electrospray Response of Peptides via Zwitterionic Additives  54th American Society for Mass Spectrometry Conference, Seattle, WA (2006)

 

·         Xiarong Liang, Harsha P. Gunawardena, Scott A. McLuckey “Electron Transfer Ion/Ion reactions of Doubly and Singly Charged Peptides: Effect of Cooling Gas Pressure” Turkey Run Analytical Conference, Marshall, IN (2005)

 

·         Joshua Emory, Min He, Harsha P. Gunawardena and Scott A. McLuckey “Investigation of Dendrimer Selectivity in Negative Ion to Positive Ion Charge Inversion Reactions  53rd American Society for Mass Spectrometry Conference, San Antonio, TX (2005)

 

·         Harsha P. Gunawardena and Scott A. McLuckey “Reagent Anions for Electron Transfer to Polypeptide/Protein Ions in the Gas Phase”  53rd American Society for Mass Spectrometry Conference, San Antonio, TX (2005)

 

·         Harsha P. Gunawardena, and Scott A. McLuckey “Top-Down Identification of Class Specific Components from Complex Mixtures: Precursor Ion Scanning of Phosphoproteins  Pittsburg Analytical Conference, Orlando FL (2005)

 

·         Harsha P. Gunawardena, and Scott A. McLuckey “Characterizing Proteins and Peptides by Electron Transfer Ion/Ion Chemistry, American Chemical Society, San Diego CA (2005)

 

·         Harsha P. Gunawardena, Ravi Amunugama, and Scott A. McLuckey “Investigations of Energy Deposition during Ion/Ion Reactions for Predicting Biomolecular Fragmentation”     52nd American Society for Mass Spectrometry Conference, Nashville, TN (2004)

 

·         Peng Pan, Harsha P. Gunawardena, Yu Xia, and Scott A. McLuckey “Selectivity of Electrospray Ionization of Proteins Solution pH and Protein pI” 52nd American Society for Mass Spectrometry Conference, Nashville, TN (2004)

 

·         Harsha P. Gunawardena, and Scott A. McLuckey “Synthesis of Protein Complexes in the Gas-Phase” 51st American Society for Mass Spectrometry 51st American Society for Mass Spectrometry Conference, Montreal, Canada (2003)

 

·         Harsha P. Gunawardena, and Brian T. Cooper “Electrostatic Contribution of Protein-SDS Binding Investigated by Charge Ladders, CE, and ESI-MS”  49th American Society for Mass Spectrometry Conference, Chicago, IL (2001)

 

·         Harsha P. Gunawardena, Childs, A. J.; Ross, C. L.; Cooper, Brian T. Cooper “Protein MEKC: Surfactant Binding and Monoclonal Antibody Analysis,” invited talk, Eastern Analytical Symposium, Atlantic City, NJ, October 2001

 

·         Harsha P. Gunawardena, and Brian T. Cooper Charge Ladder Calibration of Protein-SDS Multiple Binding Equilibrium by Capillary Electrophoresis  High Performance Capillary Electrophoresis, Boston, MA (2001)

 

·         Harsha P. Gunawardena, Minshan Shou, and Brain T. Cooper  Capillary Electrophoresis for Measuring Protein-Amphyphylic Binding Interactions  Triangle Area Mass Spectrometry Discussion Group Symposium, Research Triangle Park, NC (2001)

 

·         Harsha P. Gunawardena, and Brian T. Cooper  “Protein-SDS Binding and Signal Suppression by ESI-MS  48th  American Society for Mass Spectrometry Conference, Long Beach, CA (2000)