Proteomics and Metabolomics Core Facility
Proteomics and Metabolomics, a part of system biology, is foundational for a full understanding of a biological system including a metabolic process or disease state, and has received considerable attention since the sequencing of the human genome. Oxidative/nitrosative damage by reactive radical species appears to be central to the pathogenesis of many human diseases including cancer and is central to a broad range of biotic and abiotic stress and physiological responses in microorganisms, plants and other animals. Specific modified proteins or metabolites are generated following stressing insults and accumulate in different degenerated tissues and fluids, determining altered organ functionalities. Mass spectrometry based technologies have been propitious to the development of molecular medicine, especially in the discovery of diagnostic biomarkers of oxidative and nitrosative stress enabling early detection of diseases such as lung cancer from these biological fluids. Although Redox Proteomics and Metabolomics is currently a nascent field of research, the opportunities for mass spectrometry based biomarker discovery involved in disease strictly linked to oxidative/nitrosative stress are clear and compelling. Clinical research also hopes to benefit from proteomics and metabolomics by both the identification of new drug targets and the development of new diagnostic markers Nature (422, 115-116; 2003).
Proteomics and Metabolomics Core
Laboratory for Proteomics and Metabolomics analysis at the Redox Biology Center (RBC), Department of Biochemistry, UNL supports Faculties, scientists, clinicians and students within the RBC, University of Nebraska Lincoln, UNMC and other universities and industries. The Core provides all the tools of modern functional proteomics and metabolomics. The facility is equipped with chromatography and mass spectrometry based state-of-the-art technologies for proteomics and metabolomics (Clinical and non clinical), personalized experimental design consultation and comprehensive, individualized bioinformatics support.
We are interested in collaborations with faculties/ scientists in studying systems of biomedical importance such as clinical proteomics and metabolomics, biomarker discovery and validation, proteomic and metabolomics of plant/microbial and from other biological origin. Please contact Jiri Adamec, Core Director, regarding services provided.
All services provided are commonly used for basic biological research from biomarker discovery to target validation assay and pharmacokinetic studies.
General Services Offered by the RBC Proteomic and Metabolomic Core Facility:
- Small molecule exact mass determination or quantitation using positive or negative ion mode
- Proteomics: Protein identification using LC/MS/MS analysis and MASCOT and SEQUEST database search
- Shot gun proteome analysis of biological samples.
- Biomarker discovery from biological fluid
- Drug-protein or drug - nucleic acid - protein interaction.
- Protein complex isolation and identifying interacting partners and its quantitation.
- Protein differential expression analysis and quantitation by 2D-LC MS/MS (MudPIT).
- Global PTM analysis and quantitation
- Specialized in phosphorylation and oxidation analysis
- Coomassie Blue and Silver Stained Gel analysis
- de novo peptide sequencing by tandem mass spectrometry
- Confirmation of mutations in protein
- Customized sequence search of in-house proteins that are not available in database
- Post translational modifications (phosphorylation, sumoylation, ubiquitination, oxidation, etc.)
- Determination of oxidation state of cysteine (disulfide bonds)
- Intact proteins and peptides mass determination
Proteomic Services Available:
- Redox proteomics
- Quantitative “shot gun” differential expression/biomarker discovery proteomics (mudPIT)
- High throughput targeted SRM/MRM peptide/proteomic assay development
- Multiplex assays for biomarker research and clinical application
- Organelle proteomics
- Membrane proteomics
- Signaling pathway analysis
- Protein complex analysis
- Peptide/nucleotide/protein-ligand interactions
- Protein sequencing and identification
- Characterization of protein modifications such as phosphorylation, oxidation, methylation
- Microbial, plant, human and animal proteomics includes studies on autophagy/apoptosis, cellular dysfunction, secretome analysis
- Top-down proteomics (coming soon!)
- Antibody-based multiplexed assay (coming soon)
Discovery Proteomics: Routine shot gun, quantitative proteomics based protein expression difference analysis using MudPIT (Multidimensional Protein Identification Technology, 2-D LC/MS/MS) and multiplexed, labeled (SILAC, iTRAQ) quantitative proteome analysis from any complex biological samples (clinical or non clinical).
Targeted Proteomics: Multiple reaction monitoring (MRM) based multiplexed proteomic assay development, relative and absolute quantiation (labeled and label free) of specific proteins and peptides from plasma, serum and other biological samples.
Metabolomic Analysis Services Available:
Discovery metabolomics: UHPLC/MS based routine shot gun quantitative metabolite profiling from complex biological samples (clinical or non clinical).
Targeted metabolomics: UHPLC/MS based Multiple reaction monitoring (MRM or SRM) of metabolite, small molecules/drug, lipid assay development, relative and absolute quantiation (labeled and label free) of specific or group of metabolites from plasma, serum, urine, CSF, tissue and other clinical or non clinical biological samples.
Redox Metabolomics and Quantitative Global Analysis of endogenous metabolites from cells, tissues, fluids and whole organisms.
Instruments and Technology
The instrumentation is located in E156 of the Beadle Center. The RBC proteomic and metabolomic core Facility is equipped with 4000 QTRAP, ABSCIEX (Triple Quadrupole Ion Trap), Q-Star XL, ABSCIEX (Quadrupole-TOF) and ThermoFisher, LCQ – Fleet (ion trap) tandem mass spectrometers to provide a wide range of sample analysis. These instruments are equipped with ESI (micro spray and nanospray) and APCI sources to provide a wide range of ionization capabilities.
ABSCIEX Q-TRAP 4000 integrated with Agilent 1200 and Dionex U3000
QSTAR® XL Hybrid LC/MS/MS
LCQ Fleet integrated with 2D-nano LC/MS/MS system (ThermoFisher, USA)
LTQ Velos Pro System with ETD (Thermo Electron, USA)
Protein Bioinformatics Database:
The facility also maintains a data workstation dedicated to both qualitative and quantitative proteomics data processing, database searching, and generation of reports. Several popular software packages are installed on the data station, including Bioworks, XCalibur, Analyst, SEQUEST and Mascot server.
Metabolomic Bioinformatics Software:
Several popular software packages are installed such as Analyst, Marker View, Lipid View (AB SCIEX)
ASMS 2011, Denver, CO
N.Madayiputhiya, J. Jason, R.Nandakumar and S.M. Wells. Metabolomics: Fast UPLC/MS/MS targeted MRM analysis of L-arginine, homoarginine, ADMA and SDMA levels in mouse serum and tissue samples. 59th ASMS proceedings (2011).
USHUPO, 2011, Raleigh, NC
Renu Nandakumar, Christophe Espírito Santo, Nandakumar Madayiputhiya and Gregor Grass Quantitative label free proteomic profiling of the E.coli response to metallic copper surfaces, USHUPO (2011).
Recent Collaborative Publications:
Xu XM, Turanov AA, Carlson BA, Yoo MH, Everley RA, Nandakumar R, Sorokina I, Gygi SP, Gladyshev VN, Hatfield DL. Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery. Proc Natl Acad Sci U S A. 2010 Dec 14; 107(50):21430-4. Epub 2010 Nov 29.
Nandakumar R, Espito Santo, Madayiputhiya N, and Grass G. (2011) Quantitative proteome profiling of Escherichia coli responses to metallic copper surfaces Biometals 24:429–444.
Y. Zhu, R. Nandakumar, M.R. Sadykov, N. Madayiputhiya, T.T. Luong, R.Gaupp, Cy Lee, G.A. Somerville. RpiR homologues link Staphylococcus aureous RNAIII synthesis to the pentose phosphate pathway. Journal of Bacteriology, 2011 Nov; 193(22):6187-96.
Why Bad Immunity Genes Survive? - NSF
New efforts to fight Alzheimer’s disease: additional funds available for cutting-edge research - HHS
Sites of Interest:
HUPO (Human Proteome Organization)
National Center for Biotechnology Information (NCBI)
Metabolomics - Official Journal of the Metabolomics Society
J. proteome research
American Society for Mass Spectrometry (ASMS)
Fisher Scientific 2D LC MS
Association of Biomolecular Resource Facilities
The Human Metabolome Database (HMDB)
Plasma Proteome Institute
Proteomic analysis and Oxidative Stress
National Institutes of Health