Dr. Rama Dey-Rao

Cell and Molecular Biology, Proteomics/peptidomics, Bioinformatics, Immunology, Biochemistry, Oxidative Stress Biology,Translational& Clinical Research and Botany

2005-2009
Assistant Director: Proteomics Resources, Roswell Park Cancer Institute
Director: Dr L. Kazim

As the Biologist in Proteomics Resources, I was responsible for initiating and executing scientific research and developing new strategies in proteomics research.

Scientific:

1. Performed, analyzed and interpreted several 2D-DIGE MALDI-ToF and/or nanoLC-ESI-MS/MS (Q-ToF) quantitative proteomics analyses for investigators in RPCI.
2. Independent laboratory research implementing new cutting-edge technology: Stable isotope 16O/18O labeling of peptides by LC-MS/MS analysis, interaction of proteins with nucleotides by MALDI-ToF (recombinant human CytC with ATP), quantitative proteomics using 4-plex iTRAQ, phospho-peptide enrichment and analysis, developing alternative strategies in mass spectrometry.
3. Setting short and long term goals, objectives and plans, design and implementing evaluation systems for personnel as well as methodologies against established goals. IT issues, billing, pricing, maintenance of the Resource Web Page, record keeping, budgets, cash projections, policy implementation, SOPs (scientific and organizational).
4. Served as internal and external technical expert and provided consultation to investigators from RPCI, HWI and UB and outside before and after starting their Proteomics projects to ensure successful experimental progress and outcome. (See papers acknowledging proteomics -work done by me or under my guidance: below)
5. Established 2D-DIGE MALDI-ToF and/or nanoLC-ESI-MS/MS quantitative proteomics services including experimentation and publication.
6. Maintained and operated Waters MALDI-ToF Micro MX mass spectrometer, operated nanoLC-ESI-MS/MS (Q-ToF Premier) and related UPLC and HPLC equipment.
7. Supervised and instructed the Proteomics technologist and other RPCI personnel including students who performed experiments in the Proteomics Resources.
8. Presented the department to students, teachers, peer group and middle management group at RPCI.

Grant Application
9. Applied for a grant from American Lung association (ALA) in 2005 as Principle Investigator. TITLE OF RESEARCH PROJECT: "Novel Thiol Proteomics Approach for Protein Biomarker Discovery in Lung Carcinogenesis."
Administrative:
10. General laboratory and resource responsibilities including IT issues, billing, maintenance of the Resource Web Page, records keeping, budgets, cash projections, ordering of supplies.

1996-1999
Scientist (Training Grant) Oral Biology,
Dr M.I. Cho/Dr R. Genco SUNY at Buffalo, NY
1) Created a suppression subtractive hybridization library with odontoblast -specific enriched cDNA in E.coli.
2) Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones from the library, sequencing and studying homology.
3) Characterization of PEX.

1994-1996
Scientist, Biol. Sciences, SUNY at Buffalo, Buffalo, NY
Dr OP Bahl

1. Over expression, purification and characterization of the extracellular domain of the rat lutropin receptor (LHR) in E coli.

1992-1994
Post -Doctoral Research Associate, Biochemistry, SUNY at Buffalo, Buffalo, NY
Dr M Patel

1) Worked on tissue-specific expression of the human pyruvate dehydrogenase alpha (Pdha1)/chloramphenicol acetyltransferase fusion gene in transgenic mice.
2) Site directed mutagenesis of Pdha-1 promoter sequences to study structure function relationship.
3) Lab Manager.
Papers Acknowledging Proteomics work done

1) Drake EJ, Nicolai DA and Gulick A (2006) Chemistry & Biology 13, 1-11
2) Malinin1 NL, Zhang L, Choi , Ciocea A, Razorenova O, Ma Y-Q, et al (2009) Nature Medicine 15(3), 313-318
3) Kunapuli P., Jang G.F., Kazim L, Cowell J.K. (2009) J Mol Neurosci. Apr 23 [Epub ahead of print]

Research Skills

Mass Spectrometry and Bioinformatics
Certified training from Waters Corporation for operating MALDI-ToF and Nano-Acquity UPLC system with the Q-ToF Premier as well as Q-ToF API-US mass spectrometers.

MALDI-ToF based proteomics
* Protein ID by automated MALDI-ToF PMF (Waters)
* Protein and peptide MW determination by MALDI-ToF MS
* Peptidomics: Automated peptides profiling from biological fluids for disease biomarkers discovery using MALDI-ToF MS together with statistical analysis

Q-ToF based proteomics
* Protein and peptide MW determination by ESI-MS and LC-MS
* Protein ID by LC-MS/MS (UPLC and Q-ToF, Waters) form 1D gel bands and 2D gel spots
* Protein mixture ID by LC-MS/MS (in-solution digestion)
* ID of Protein-protein interaction from immunoprecipitated sample using 1D gel
* Post-translational modifications (PTMs) characterization (phosphorylation, oxidation, ubiquitination, etc)
* 16O/18O-labeling technique for protein C-terminal determination: experimental design, sample preparation as well as analyses. Developed method on standard protein
* Manual De novo sequencing and data interpretation

Gel based proteomics with DIGE (Differential in Gel Expression)
* Experimental design, sample preparation and labeling with CyDyes (Cy2, Cy3 and Cy5) and 2D gel electrophoresis
* Statistical analysis for 2D- DIGE data (T-test and ANOVA)
* Image acquisition by Typhoon 9410 and image analysis and protein quantitation by Decyder software
* Automated sample handling by auto spot picker and digester and protein identification by MALDI-ToF and LC-MS/MS

Bioinformatics
* Software for analysis of MS and MS/MS data + Database search engine (Masslynx, Proteinlynx MaxEnt 1 and 3)
* Statistical analysis for 2D- DIGE data (T-test and 1-way and 2-way ANOVA)
* Algorithms for metabonomics (R, XCMS and MZmine2, MeV)
* De novo sequencing for lC-MS/MS data &Search engines etc (Mascot, PLGS, X! Tandem, Peaks, Scaffold)
* Gene annotation: (GeneOntology, Entrez)
* Proteomics software tools: NIH databases, KEGG database, Metline database, BLAST, Unimod, Peptide Atlas, Phosphosite, Metacore, Panther, Protein Prospector, all available proteomics tools from ExPASy etc.
* Partek Genomic Suite for interpreting Microarray data (Affymetrix HG_U133_Plus-2), GeneSifter, Ingenuity Pathway analysis, TMeV, Cytoscape, BioMart, DAVID, Gsea etc.

Molecular Biology
* cDNA library construction by suppressive subtractive hybridization, screening, sequencing, BLAST searching for homology, annotation.
* Gene expression by Microarray using Affymetrix Genechip HG_ U133_ Plus2.
* Gene cloning, subcloning, restriction analysis, sequencing, hybridization (Southern and Northern blots), isolation of nucleic acids, in-vitro translation, screening of cDNA libraries.
* Transgenic techniques in mice (all aspects of animal handling, operations, screening by PCR) Tissue assays for CAT expression, breeding to generate stable transgenic lines
* Site directed mutagenesis, PCR, RT-PCR, quantitative real time PCR, tissue culture of transfected and primary cell lines (mammalian cell culture), CAT assays
* DNase/P1 nuclease hypersensitivity assays.
* DNA sequencing, DNA-protein interaction analysis (gel-retardations(gel shift assays, ChIP assay, in vivo and in vitro DNase footprinting, and methylation interference assay)
* Extensive use of software from NIH for genomics and molecular biology.
Immunology/Immunocytochemistry
* All aspects of preparing antigen, injection into animals, collecting blood or polyclonal antibody from egg albumin, all aspects of ELISA, Westerns, characterization and identification of antibodies.
* Indirect immunoflourescence, BrdU and biotin-streptavidin labeling replicating DNA, PAP immunocytochemistry.
* Cytology: staining (nuclei, chromosomes, and nuclear bodies), COMET assay
* Fluorescence microscopy, photography developing etc.
Protein Chemistry
* Electrophoretic analysis of Proteins, quantitations, Western blots, silver and Coomassie blue, Deep purple staining of standard 1-D and 2-D gels.
* Specialized in purification of proteins by gel filtration, Ion exchange, Affinity, reverse phase chromatography, FPLC, HPLC, UPLC, Spectrophotometry
* Polyclonal antibody production and purification, ELISA. Expression, purification and characterization of recombinant protein in E.coli.
* Radioimmunoassays, ligand binding assays, receptor binding assays
Analytical Chemistry
* Analytical tests involving Lipid Peroxidation, Vitamin profiles, LHP profiles, HPLC analysis of DNA damage, and several enzyme assays

Culture Techniques
* Animal tissue (primary) culture techniques, Bacterial cell culture, Yeast cell culture, Mammalian cell-line culture.

Electron microscopy
* Transmission Electron Microscopy (fixing, embedding, ultramicrotomy, developing and printing micrographs.
Selected Publications and Posters

1) Dey-Rao R., Beachy, S.H., Choi,.K.S., Jang, G-F., Repasky, E.A., Kazim, A.L. (2008) Discovering serum protein markers for tumor detection and monitoring therapeutic response in a Human tumor-SCID mouse model. Journal of Biomolecular Techniques 19(1): P161-S, 51-52.

2) Park JC, Park JT, Son HH, Kim HJ, Jeong MJ, Lee CS, Dey R, Cho MI. (2007) The amyloid protein APin is highly expressed during enamel mineralization and maturation in rat incisors. Eur J Oral Sci. 2007 Apr; 115(2):153-60.

3) Eun-Mi Park, Kyoung-Soo Choi, Soo-Yeon Park, Sun-Hee Baek, Rama Dey-Rao, Haitao Zhang, Yeul Hong Kim, Jong Hoon Park, Clement Ip, and Young-Mee Park "(2006) Analysis of protein redox modification by hypoxia" Special Proteomics Issue of Preparative Biochemistry and Biotechnology. 36:65-79.

4) Jackson LW, Schisterman EF, Dey-Rao R, Browne R, Armstrong D. (2005) Oxidative stress and endometriosis. Hum Reprod. 20(7) 2014-20

5) Dey R., Son HH, Cho MI, (2001) Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive hybridization. Arch Oral Biol. 46(3): 249-60.

6) Podgornaya O, Dey R., Lobov I, and Enukashvili N (2000) Human satellite 3 (HS3) binding protein from the nuclear matrix: isolation and binding properties. Biochim Biophys Acta. 1497(2): 204-14.

7) Dey R., Naik S., Patel MS. (1996) Tissue-specific expression of the human pyruvate dehydrogenase alpha (Pdha-1)/chloramphenicol acetyltransferase fusion gene in transgenic mice. Biochim Biophys Acta. 1305(3): 189-95.

8) Nair S, Dey R, Sanford JP, Doyle D. (1992) Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein. J Biol. Chem. 267(18): 12928-35.

9) Poltaratsky V.P., Dey R., Belgrader P, Berezney R. and Podgornaya O (1991) proteins bound to satellite DNA are present in human nuclear matrix cell preparations Molecular Biology 25, 83-90

10) Belgrader P, Dey R. and Berezney R. (1991) Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain. J Biol Chem. 266(15): 9893-9.

Oral Presentations , Seminars and Workshops Conducted

* Hylander B., P. Liang, Rama Dey-Rao, Gibbs J., Pitoniak R., Sirota, I., C-T. Lee, E.A. Repasky (To be held Nov, 2009) Defining the anti-tumor efficacy of Apo2L/TRAIL against patient pancreatic tumor xenografts in SCID mice. NCI Translational Meeting, Washington DC.
* Coordinated with Waters Corp and Applied Biosystems to hold 2 free educational seminars at RPCI, 2006-2007
* Was actively involved in organizing a 2 day workshop in August 2008, with Dr Jeremy Nicholson (Imperial College, London) and group with oral presentation.
* Was jointly responsible for inviting Dr Josep Villanueva (MSKCC, NYC) for a 2 day workshop in Sept 2008 along with the department of Molecular and Cellular Biophysics and Biochemistry.
* Dey R., Cho MI. (1999) Characterization of a 1.7-kb 3' extension of PEX cDNA in rat odontoblast. Journal of Dental Research 78, Abstract 134, pp122. Oral presentation at AADR meeting in Vancouver

Selected Published Abstracts and Posters

1. Kazim, A.L., Dey-Rao, R., Jang, G-F. (2008) Proteomics Resources. Journal of Biomolecular
Techniques 19(1): P139 (F)-M, 44.
2. Dey-Rao R, Jang GF: Proteomics. (11/2007) Northeast Regional Life Sciences Core Directors
Meeting, Poster, Ithaca, NY
3. Dey-Rao R, Jang GF: Proteomics. (11/2006) Northeast Regional Life Sciences Core
Directors Meeting, Poster, Ithaca, NY
4. Dey R., Bahl, OP (1996) Overexpression, purification and characterization of the extracellular domain of the rat lutropin receptor in e coli. Poster session of the 10th International Congress of Endocrinology June 12-16

RESEARCH STATEMENT
Rama Dey-Rao, PhD

I have acquired a broad and diverse training from my past and present research experience that makes me a highly competitive candidate for consideration as an independent scientist or working within a group. It demonstrates my ability to be a good collaborator, team player and mentor to students. Overall, my experience has contributed to a "systems biology-like" approach in my career which allows me to effectively communicate between biologists, chemists, computer scientists, programmers and statisticians.

Below are examples of my previous research experience as well as the different -omics platforms that I have used and collaborations that illustrate my diverse background in biological sciences.
Proteomics Resources, Roswell Park Cancer Institute 2005-2009
Proteomics I established the DIGE-based quantitative proteomics platform in the facility. Several comprehensive DIGE- MALDI-Tof and LC-ESI-MS/MS based quantitative proteomics projects were undertaken in collaboration with many investigators at RPCI, SUNYAB and HWI. Working with Y. Rustum's group, differential protein expression between Selenium treated and untreated Mice with A549 tumors as well as A549 cells in culture was studied by DIGE followed by Decyder analysis and protein identifications by mass spectrometers. Using serum from a human tumor -SCID mouse xenograft model in collaboration with Repasky Lab (Immunology, RPCI), we studied the in vivo differentially expressed proteins correlated with the presence or absence of a colon adenocarcinoma (Colo 205) as a baseline using both DIGE- and the "Dilute and Shoot" MALDI-ToF MS technology. We studied the response of the established tumor to Apo2L/TRAIL (death ligand of the TNF family -Tumor necrosis factor) and found that the up-regulated serum proteins characteristic of acute phase response in mice bearing tumors did not show the same trend after the treatment. A single protein Fibrinogen beta chain precursor was found to be decreased by 3 -fold in the treated samples. This identification was confirmed by nano-LC-ESI-MS/MS analysis. [1].

Microarray Collaborating with B. Hylander in Repasky Lab (RPCI), I am consolidating genechip expression data by microarray (Affymetrix HG-U133_Plus2 platform) from patient pancreatic tumors in order to identify genes that are differentially expressed in tumors which are sensitive and those which are resistant, to Apo2L/TRAIL treatment. The goal of this project is to predict the response of pancreatic tumors to therapy. This high-throughput method helps to monitor RNA or DNA for mRNA expression. The work is being compiled for publication.

I have attended a 2-day workshop "Interpreting gene-lists from -omics studies (July 9-10, 2009) from the Canadian Bioinformatics Workshop (Ontario Institute of Cancer Research). This implemented experience will enhance the capacities to successfully handle large sets of data generated by microarray, genomics proteomics and metabonomics, sort by bioinformatics tools and interpret the biological significance.

Metabonomics I am participating in an on-going cross-departmental translation study on biomarker discovery in renal cell carcinoma between Mass Spectrometry facility at RPCI, NMR facility at UB and Dept of Biostatistics at UB. Metabolites are the end products of an organism's biological activity and are readily secreted into the serum. A cross-platform metabonomic profile using both NMR and LC-MS is being subjected to statistical analyses to remove systematic bias inherent in -omics based methods and identify spectral features of metabolites that can discriminate between diseased and normal state in patient serum samples.

Peptidomics I have performed detailed "proof of principle" studies on "peptidomics" using methods described by the Tempst group at Sloan Kettering Cancer Center. Using a manual Dynabeads (RPC18) procedure, along with MALDI-ToF MS I was able to separate the non-polar peptides from normal human serum and identified a ladder of peptides from one of the abundant proteins, as has been shown by Villanueva. The expectation is that a diseased sample shows variations in the pattern of peptide ladders generated ex-vivo due to the presence of disease specific exo-proteases. Villanueva et al (2006) [2] described a set of 61-disease specific peptides that could separate between controls and 3 clinical groups, prostate, bladder and breast cancer. These surrogate markers were generated ex-vivo in serum and not plasma which could be explained by the presence of disease specific proteases acting on the abundant protein peptides generated in the clotting process of serum. Working with Robert Leach (scientific programmer, CCR, SUNYAB), we have developed a pipe-line for the handling the MALDI-ToF MS data (criteria for normalization, background subtraction and peak alignment towards statistical validation of relevant discriminatory peaks).

Technical projects I took on several proteomics technical projects which have the potential to be used in any project requiring similar methodology: Working for the Nikiforov lab (Cell Stress Biology, RPCI) I have established the 16O/18O-labeling technique for protein C-terminal end determination. Enrichment and characterization of several post-translationally modified peptides (oxidation, phosphorylation. sulfation, ubiquitination etc) have been undertaken. These techniques allow investigators to follow up on regulatory and mechanistic studies after identifying key protein components in signaling pathways.
International Collaboration 1990-2009: I have collaborated with Dr O.I. Podgornaya (Institute of Cytology, Russia) from 1989, dealing with HS3 (Human satellite-3) binding proteins (~70kDa) in the nuclear matrix. [3, 4] It appears now that satDNA is involved in nuclear 3-D organization and in some processes such as ageing, carcinogenesis, response to cell stress and probably early embryogenesis thus changing the definition of satDNA as DNA representing a special type of highly condensed, transcriptionally silent, constitutive heterochromatin. Nano-LC-ESI-MS/MS analysis of proteins run on 1-D gels was undertaken by me in a special collaboration with the Podgornaya lab. Details of de novo sequenced peptides along with extensive library searches (including a specialized database) are helping her identify novel proteins (not in the public databases) from Halisarca dujardini. Olga has shown a keen interest in collaborating on joint grant submissions which can and will allow free exchange of ideas and collegiality. (Attached: invitation to visit Russia, 2009)

Translation Research in IMMCO Diagnostics and Zeptometrix Corp. 2000-2004: My experience in translational research taking bench science to the market is an invaluable asset to graduate and post doctoral students. Using my expertise in molecular cloning, protein purification and characterization, I cloned and purified to >90% IMMCO Diagnostics' first recombinant protein: HSP-70 used for a Western Blot Assay Kit for the autoimmune disease, sensorineural hearing loss (SNHL). While setting up the entire "Oxidative Stress Program" as Director, (Zeptometrix Corp.) in collaboration with Harold Box (Cell Stress Biology, RPCI) and Richard Browne (Biotechnical And Clinical Laboratory Sciences, UB) I launched 4 Assay kits (total 14 products) for analysis of oxidative stress levels from human serum, including QA/QC controls setup, pricing, advertising and marketing. I served as the technical lead on new product development and technical expertise on multiple projects. Novel concepts and strategies were adopted to quickly develop a palette of kits and assays for oxidative stress burden measurements. Within 2 years I had 14 products on the markets with scientific and market research completed along with marketing strategies, fliers, targeting customers and introduction is SFRBM (Society for Free radicals in Biology and Medicine) annual national meeting for 2 years. I started and gave the vision for a service department (OXI-test) and successfully completed several contracts for clinical research. [5]

Department of Oral Biology (University at Buffalo) 1997-2000 As a first step to better understand the regulatory mechanisms responsible for odontoblast differentiation and dentinogenesis, I cloned and characterized genes from an odontoblast-specific/enriched rat cDNA library (>75%enriched) created by suppression subtractive hybridization. [6] With support from USPHS grant DE 07034 and DE 4849, working in the Department of Oral Biology, SUNYAB. I established the basis for continuing research and subsequent characterization of AP1 indicating a functional role in mineralization and maturation of enamel. [7]

Summary: I have experience in cell and molecular biology, protein expression, protein purification, immunology, proteomics, bioinformatics, metabonomics, peptidomics, gene expression microarray (Affymetrix platform), bioinformatics and systems biology. This diversity in my experience allows me to work well within a dynamic group of scientists.



Reference:

[1] Dey-Rao R., Beachy, S.H., Choi, K.S., Jang, G-F., Repasky, E.A., Kazim, A.L. (2008) Discovering serum protein markers for tumor detection and monitoring therapeutic response in a Human tumor-SCID mouse model. Journal of Biomolecular Techniques 19(1): P161-S, 51-52.
[2] Villanueva J., Shaffer D.R., Philip J., Chaparro C.A., Erdjument-Bromage H., Olshen A.B., Fleisher M., Lilja H., Brogi E., Boyd J., Sanchez-Carbayo M., Holland E.C., Cordon-Cardo C., Scher H.I., Tempst P.(2006)Differential exoprotease activities confer tumor-specific serum peptidome patterns. J Clin Invest Jan; 116(1):271-84.
[3] Podgornaya O., Dey R., Lobov I., and Enukashvili N. (2000) Human satellite 3 (HS3) binding protein from the nuclear matrix: isolation and binding properties. Biochim Biophys Acta. 1497(2): 204-14.
[4] Poltaratsky V.P., Dey R., Belgrader P., Berezney R. and Podgornaya O. (1991) proteins bound to satellite DNA are present in human nuclear matrix cell preparations Molecular Biology (Translated) 25, 83-90.
[5] Jackson L.W., Schisterman E.F., Dey-Rao R., Browne R., Armstrong D. (2005) Oxidative stress and endometriosis. Hum Reprod. 20(7) 2014-20.
[6] Dey R, Son H.H., Cho M.I. (2001) Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive hybridization. Arch Oral Biol. 46(3): 249-60.
[7] Park JC, Park JT, Son H.H., Kim H.J., Jeong M.J., Lee C.S., Dey R., Cho MI (2007) The amyloid protein APin is highly expressed during enamel mineralization and maturation in rat incisors. Eur J Oral Sci. 2007 Apr; 115(2):153-60.


BIO2010: The National Academies advises the Nation on Science, Engineering, and Medicine education.

2002-2004
Program Director: "OXIDATIVE STRESS", Zeptometrix Corp.
CEO: Dr J. Hengst

Responsible for starting a new Division "Oxidative Stress" at Zeptometrix Corp.

Scientific:
1) Performed all research and tests for TBARS, 8-F2-Isoprostane, Paraoxonase activity, Vitamin A and E and Lycopene for "Endometriosis" project (Jackson LW, Schisterman EF, Dey-Rao R, Browne R, Armstrong D. (2005) Oxidative stress and endometriosis. Hum Reprod. 20(7) 2014-20)
2) Provided scientific and administrative leadership on 14 new kits/products (analytical chemistry and immunochemistry-ELISA tests) dealing with analyzing oxidative stress burden (e.g. TBARS, Glutathuione peroxidase and reductase, lipid profiling, genomic DNA kit) Initiating, SOP generation, pricing, support and production as well as marketing.
3) Conducted/completed several clinical research projects on assessment of oxidative stress burden in patients with endometriosis etc., with the launch of a service department under the name "OXI-test."
Grant Application:
4) Applied for STTR Phase I grant as commercial partner Co-PI, Phase 1 grant (2004) with Dr. Harold Box, Dr. Rodabaugh (Roswell Park) and Dr. James Olson (UB) with the title "Products and Services for Evaluating Anticancer Drugs."

2000-2002
Director: Polymer Biosciences, ImmcoDiagnostics, Buffalo, NY
Owner: Dr Vijaykumar

As head and scientific lead of the Polymer Biosciences Facility at ImmcoDiagnostics Inc. a biotech company dealing with autoimmune diagnostic products and services, I performed entire research behind several projects:

Scientific:
1) Cloning and purification of the company's first recombinant protein HSP-70 which was used by the company in one of their most highly used diagnostic tests for autoimmune hearing loss disease.
2) I was responsible for initiating, directing and executing scientific research and/or development strategies using the established chromatographic facility BioCad 700 (ABI) (HPLC), and molecular biology facility (established by me). All technical problems and issues were solved by me and I fielded all customer questions on my products.