The mission of the Proteomics Core in the Department of Pharmacology and Experimental Neuroscience is to provide state-of-the-art scientific and technical knowledge in proteomics, to facilitate and enhance on-going research projects, and to promote the application of proteomics technologies in the future studies. To accomplish these goals, the Proteomics Core assists in proteomic profiling of differentially expressed proteins, proteins identification and characterization, and in training, education and consultation in this rapidly developing scientific field.
Successful completion of sequencing of the human genome showed considerably smaller number of genes (20,000 to 25,000 protein coding genes) (International Human Genome Sequencing Consortium Lander ES, 2004) than expected and the number of proteins greatly exceeding the number of genes. This had a big impact on how we envision human and other proteomes and how we approach proteomic analysis. Isoforms, products of one gene modified by posttranslational modifications or alternative splicing, is only one part of complexity because function and biological role can also depend on localization, cytoplasmic, in sub-cellular compartments, and/or extracellular.
Interactions with other proteins and non-proteinacious molecules add yet another layer of complexity. Proteomics as a scientific field emerged in mid-1990 along with technological advances in analyses of proteins and peptides. Two-dimensional polyacrylamide gel electrophoresis (2D SDS-PAGE) originally developed in late 70’s (Barritault et al., 1976) was rapidly advanced to the next level when immobilized pH gradient (IPG) strips (Gelfi and Righetti, 1983) facilitated analysis of gene products containing over 2000 proteins in a mixture (Gianazza, 1995) with much higher reproducibility were introduced.
Mass spectrometry of proteins and peptides is developing fast providing new methods and reagents for quantitative analysis such
Isotope Coded Affinity Tags (ICAT). Protein microarrays, recently emerged, adding yet another powerful tool in global screening of gene products in complex biological systems.
Development of user friendly kits for protein sample preparation and extraction enabled non-protein chemists to isolate proteins, fractionate complex samples and develop their own protocols in a similar way as development of expression vectors and systems (Feuerstein et al., 2005).
Growth of all kind of databases, new and improved algorithms for database searches, opened the door for high throughput experiments with substantially increased quality of protein identification and quantitative measurements (Ku and Yona, 2005).
It became a reality to identify fingerprints of changes: induced by infection, malignant transformation, exposure to toxic agents, or differentiation (Pang et al., 2006). Substantial progress in prognosis and monitoring of disease progression and discovery of new biomarkers and drug targets is widely expected.
Our approach to proteomic analysis consists of four steps: fingerprint, sample fractionation, protein identification, and data analysis.
Fingerprinting. Two technologies are used: Surface Enhanced Laser Desorption Time-of-Flight (SELDI-TOF) mass spectrometry and 2 dimensional polyacrylamide gel electrophoresis (2DE) with DIGE technology. Two systems for 2DE analysis are available, ProteanIIxi (BioRad, Hercules, CA) and Ettan Dalt Six (Amersham/GEHealthcare, MA).
|SELDI-TOF Workstation is designed to perform mass spectrometric analysis of protein mixtures retained on chromatographic Protein Chip® surfaces. This instrument produces spectra of complex protein mixtures based on the mass/charge ratio of the proteins in the mixture bound to the surface of specific chip. Differentially expressed proteins may be determined from these protein profiles by comparing peak intensity.|
|BioRad Protean IEF and Protean IIxi|
|Amersham/GE Healthcare System|
|Example of 2DE DIGE of CSF samples from HIV-1 infected individuals with or without cognitive impairment (HAD). Green spots (Cy3) are from HAD-negative, while red spots (Cy5) are from HAD patient.|
Protein fractionation. State of the art HPLC system from Shimazu, Inc, and Akta FPLC system from AmershamPharmacia, Inc are available for protein fractionation and purification. Broad variety of columns are available, e.g., Multiple Affinity Removal Column (Hu-6HC) from Agilent Technologies for removal of six most abundant proteins (human serum albumin, IgG, a1-antitrypsin, IgA, transferring, and haptoglobin) from serum, CSF, and culture supernatants samples.Protein fractionation. State of the art HPLC system from Shimazu, Inc, and Akta FPLC system from AmershamPharmacia, Inc are available for protein fractionation and purification. Broad variety of columns are available, e.g., Multiple Affinity Removal Column (Hu-6HC) from Agilent Technologies for removal of six most abundant proteins (human serum albumin, IgG, a1-antitrypsin, IgA, transferring, and haptoglobin) from serum, CSF, and culture supernatants samples.
|Protein identification. We are equipped with a state-of-the-art Ion Trap Mass Spectrometer interfaced with a 2-dimensional Liquid Chromatography System, a ProteomeX LCQDecaPlus (ThermoFinnigan, Inc). The instrument allows peptide sequencing to be performed either in a manual mode (sample is sprayed from metalized capillary at the level of 100 nl/min) or in a fully automated mode (sample is injected by autosampler onto chromatographic column and eluted by applied gradient also at the flow rate of 100 nl/min). These configurations allow analysis of either complex mixtures of peptides or in depth analysis of a single peptide. The latter is important for uncovering post-translational modifications such as glycosylation, acetylation, phosphorylation, formylation, oxidation etc. Phosphorylation can also be detected either in a manual mode or in an automated mode using a “neutral loss" setting of MS/MS. All together we have the unique capability to analyze proteolytic digests of complex mixtures at a very low level, such as, those from tryptic digests of single spots from 2D SDS-PAGE tests. In support of this notion, our nano-spray LC-MS/MS system requires as little as 10-20 femtomoles of peptide for detection.|
Data analysis. Our primary source of protein information is NCBI fasta.nr database. The reason for this choice is that BioWorks 3.1SR software, which is integral part of ProteomeX LC-MS/MS system uses this database. We are, however, not limited to this source. Other sources such as Swiss-Prot and TrEMBL are also available and are in public domain. All other sources for post-translational modifications etc. are also available through the ExPaSy Tools web site. Database searches are performed on two computer stations. Proteomics Core has designated storage space on the 2 terabyte server. Statistical analysis is supported by Dr. James Anderson and Mr. Fred Ullrich from the Department of Preventive and Societal Medicine at the University of Nebraska Medical Center.
1 R21 MH075662-01 PI: Pawel Ciborowski
"Macrophage Protein Fingerprints in HIV-1 Dementia"
R37 NS36126 PI: Howard Gendleman
"Blood-brain Barrier Physiollogy and HIV Dementia"
P01 NS43985 PI: Howard Gendelman
"Neural Immunity in HIV Dementia"
R20 RR 15635-02 PI: Charles Woods
"Cellular Mechanisms for HIV-1 Induced Neuronal Injury"
"Protein Structure and Function, Proteomics"
- P. Ciborowski and H.E. Gendelman. Human Immunodeficiency Virus-Mononuclear Phagocyte Interactions: Emerging Avenues of Biomarker Discovery, Modes of Viral Persistence and Disease Pathogenesis. Current HIV Research 2006, 4(3):279-91
- Y. Enose, C. Destache, A. Mack, J. Anderson, F. Ullrich, P. Ciborowski, and H. Gendelman. Proteomic Fingerprints Distinguish Microglia, Bone Marrow and Spleen Macrophage Populations. Glia. 2005, 51:161-172
- K.A. Carlson, P. Ciborowski, C. Schellpeper, T. M. Biskup, R.F. Shen, X. Luo, C. J. Destache, and H.E. Gendelman, Proteomic fingerprinting of HIV-1-infected human monocyte-derived macrophages: A preliminary report. J. Neuroimmunol. 2004, 147:35-42
- Wojna, V., Carlson, K.A., Luo, X., Mayo, R., Melendez, L., Kraiselburd, E., and Gendelman, H.E. Proteomic fingerprinting of human immunodeficiency virus type 1-associated dementia from patient monocyte-derived macrophages: A case study. J. NeuroVirol. 2004, 10:74-81
- P. Ciborowski, Y. Enose, A. Mack, M. Fladseth, and H. Gendelman. Diminished matrix metalloproteinase 9 secretion in human immunodeficiency virus infected mononuclear phagocytes: modulation of innate immunity and implications for neurological disease. J. Neuroimmunol 2004, 157:11-16
- Luo, X., Carlson, K.A., Wonja, V., Mayo, R., Biskup, T., Stoner, J., Anderson, J.,Gendelman, H.E., and Melendez, L.M. Macrophage proteomic fingerprinting predicts HIV-1 associated cognitive impairment. Neurology 2003, 60(12):1931-7