Laboratories The Pepper Center at UTMB
The Biorepository and Mass Spectrometry Laboratory (2,800 sq ft space) is located on the 10th floor of the Medical Research Building. It has a convenient small satellite laboratory (approximately 300 ft) for initial processing of human samples located on the 4th floor of the John Sealy Annex Building, one floor below the Institute for Translational Sciences-Clinical Research Center. The OAIC Biorepository and Mass Spectrometry Laboratory is directed by Dr. Elena Volpi [email]. Dr. Ming Zheng [email] is the mass spectrometry specialist.
The laboratory includes the equipment listed below:
- Four Agilent Technologies 6890 Series III Plus Gas Chromatograph 5973N Mass Spectrometers equipped with EI/PCI/NCI bundle, FID, and autosampler. These are the primary instruments to measure plasma and intracellular free amino acid enrichment.
- One Finnigan Delta Plus Gas Chromatograph-Combustion-Isotope Ratio Mass Spectrometer with GasBench which is used for the measurement of very low enrichments of muscle protein-bound amino acids.
- Three Speed-vacuum systems for sample concentration and desiccation.
- One Microcentrifuge (Sorvall).
- One Nitrogen evaporator (N-Evap) for sample desiccation.
- One Jouan bench centrifuge to separate blood subfractions.
- One Spectrophotometer.
- One MiniProtean electrophoresis system with blotting apparatus.
- Four -80° C freezers, one -20° C freezer, two 4° C refrigerator for sample and reagent storage
- One 121 L liquid nitrogen dewar for tissue sample storage
- One Biosafety sterile cabinet for tracer preparation.
- One fume hood.
- One glucose analyzer (YSI).
- One Total Nitrogen Analyzer (Cosa Instrument Co. Houston, TX).
- Standard lab items (e.g., water-baths, microplate reader, rotators, shakers, sonicators, high-speed and micro centrifuges). This laboratory provides the following services:
- Sample biorepository and tracking
- Measurement of stable isotope enrichment in biological samples by mass spectrometry (protein and glucose metabolism)
- Measurement of total nitrogen excretion
- Metabolic modeling
- Glucose and lactate analysis
- Measurement of hormones and cytokines by ELISA
The Skeletal Metabolism and Biology and Metabolism Laboratory (2,500 sq ft space) is located on the 10th floor of the Medical Research Building, adjacent to the Biorepository and Mass Spectrometry lab. It is directed by Dr. Blake Rasmussen [email], Leader of the Metabolism and Biology Resource Core and supervised by Dr. JunFang Hao [email].
This laboratory provides the following services:
- Microscopic and morphological analysis of skeletal muscle by immunohistochemistry
- Analysis of cell signaling and protein expression by SDS PAGE and immunoblotting
- Analysis of gene expression including amino acids transporters and microRNA by real-time qPCR
- Primary myotubes culture from human skeletal muscle biopsy samples
- Hormone and cytokine assay by ELISA Equipment:
- Four Bio-Rad and two Amersham gel electrophoresis systems with blotting apparatus for protein separation and western blotting.
- One Bio-Rad digital chemiluminescence gel imaging system for measurement of intracellular protein content and phosphorylation status of key proteins the regulate translation initiation and protein synthesis.
- One Cryostat.
- One Zeiss Digital Fluorescent microscope that will be used to assess changes in muscle fiber cross-sectional area due to aging and inactivity.
- One Bio-Rad CFX Connect and one iQ5 Real Time PCR that will be used to determine muscle gene expression.
- One MyCycler BioRad thermocycler.
- One Agilent BioAnalyzer.
- One ThermoFisher Scientific Nanodrop 2000 Spectrophotometer.
- One cell culture room including biosafety cabinet, incubators, inverted Zeiss microscope, Bio-Rad electroporator, cell counter, cryosystem and autoclave.
- Two -80°C freezers, two -20°C freezers, two research refrigerators.
- Two fume hoods.
- Standard lab items (e.g., water-baths, microplate reader, rotators, shakers, spectrophotometer, liquid scintillation counter, high-speed and micro centrifuges)
The Shriners Hospital Metabolism Unit performs laboratory analyses of all plasma, muscle and adipose tissue samples. The Metabolism Unit is on the 6th floor of the Shriners Hospital for Children - Galveston. (approximately 6,500 sq ft). It is directed by Dr. Labros Sidossis [email] and provides the following services:
- Measurement of stable isotopic enrichment in biological samples by mass spectrometry (fat and energy metabolism)
- Metabolic modeling
- Mitochondrial respirometry
- Three Oroboros, Oxygraph-O2k, high-resolution respirometers
- One Biotek Eon spectrophotometer
- Three Glucose/Lactate Autoanalyzers (Stat 2300, Yellow Springs Instruments, Yellow Springs, OH)
- Three Centrifuges
- Two Beckman Ultra-Centrifuges (Beckman Coulter, Fullerton, CA)
- One Bio-Rad and two Amersham gel electrophoresis systems with blotting apparatus
- One Bio-Rad digital chemiluminescence gel imaging system
- One Cryostat
- One Bio-Rad iQ5 Real Time PCR
- Two Sampler Concentrator Systems (Speed-Vac)
- Two Nitrogen Evaporators (N-Evap, Organomation Associates, Berlin, MA)
- Four -80°C freezers, three -20°C freezer and one walk-in 4°C refrigerator
- One Biosafety sterile cabinet for tracer preparation • One fume hood • Four computers (Dell Precision T3500 CMT with 22-inch wide screen Flat Panel monitor) • Numerous standard lab items (water-baths, microplate reader, rotators, shakers, spectrophotometer, etc.)
- One Delta V Isotope Ratio Mass Spectrometer (GC-C-IRMS; for analysis of carbon, nitrogen, hydrogen, and oxygen)
- Six Agilent GC-MSs: 6890 Series GC System-5973 Mass Selective Detector equipped with EI and PCI functions and one Agilent GC-MS: 7890 Series GC System-5975 Mass Selective Detector with EI function
- One network gas chromatograph flame ionization detection: (GC-FID 6890; Hewlett-Packard / Agilent Technologies, Palo Alto, CA)
- One Liquid Chromatograph Mass Spectrometer (LC-MS; Agilent Technologies)
Biomolecular Resource Facility (BRF)
The BRF is a UTMB designated core facility providing research support targeted to the analysis of biomolecules, especially proteins and peptides. The initial organization of the BRF began in 1975 and the facility has grown considerably over the years to meet the analytical needs of biomedical investigators. Dr. Alexander Kurosky, has been the Director of this facility since its inception. The BRF is currently composed of six functional cores organized into two sections, Protein Chemistry and Proteomics. In addition, the BRF is closely aligned with the UTMB Bioinformatics Program directed by Dr. Bruce Luxon, Professor in the Department of Biochemistry & Molecular Biology. Dr. John E. Wiktorowicz, the BRF Director of the Proteomics Section, Professor in the Department of Biochemistry & Molecular Biology, is a recent addition to the BRF and was formerly Director of Proteomics at Lynx Therapeutics, Inc.Biomolecular Resource Facility Website
The overarching philosophy of the BRF is to provide UTMB researchers with a menu of services encompassing state-of-the-art analytical capabilities and technologies at reasonable costs that will enhance their funded research projects as well as the quality of their publications. Investigators are encouraged to interact closely with BRF personnel to obtain maximum benefit of the BRF services. Technologies and the available instrumentation in the BRF are at the cutting edge of research. The BRF is committed to provide expert analyses, and Core personnel have been well-trained and are highly skilled in their analytical duties. In order to ensure quality of services, the BRF Cores participate in the ABRF's annual analytical testing evaluations using identical samples distributed to all member facilities. The institutional core laboratories are available to OAIC investigators. They will be used by PES1 and PES3.
Proteomics Core: In close collaboration with investigators, the Proteomics Core utilizes the highest degree of professional expert analyses and state-of-the-art technology.
Separation Services: The Separations Technology section ensures quality through the use of automated robotics, high-performance bioinformatics with robust statistical menus, and cutting-edge chemistries. In addition, this Core is engaged in developing new separations chemistries and instrumentation in order to address the detection and quantification of low abundance proteins.
Services available are:
- HPLC sample fractionation (HIC-proteins; RP-peptides)
- Preparative IEF fractionation
- Sub-cellular fractionation
- IEF, 1D, and 2D gel electrophoresis with Coomassie or fluorescence staining (Sypro-Ruby for total protein or Pro-Q Diamond for phosphorylated proteins)
- Image analysis of stained protein spots in gels
- Robotic protein spot picking from gels
- Robotic protein digestion, processing, and MALDI target application
- Training in specific applications of separations technology
Quality Control. All fractionation steps and separation media undergo extensive validation in order to ensure reproducibility. Internal standards are employed wherever possible. All quantification chemistries and instruments are certified to produce accurate and precise values. We apply sophisticated statistical tools, available in the Proteomics Bioinformatics Core to evaluate the quality of our data. Protocols and methods are shared with investigators.
Major Core Equipment:
- Beckman Optima TLX Table-top Ultracentrifuge with swinging bucket and fixed-angle rotors
- Applied Biosystems 270A-HT Capillary Electrophoresis System
- Protein ProFiler™ Liquid Two Dimensional Electrophoresis System (In Development)
- Invitrogen ZOOM Â® IEF Fractionator for preparative IEF
- Nonlinear Progenesis SameSpots with Statistics Toolbox 2D analysis software
- Agilent 1100 HPLC with diode-array detector
- Multiple 1D and 2D gel electrophoresis apparatus
- Digilab Genomic Solutions Pro-Pic II (robotic gel spot picker)
- Digilab Genomic Solutions Pro-Prep (robotic gel plug digestion)
- Fuji FLA-5100 Fluorescence Scanner
- Perkin-Elmer ProEXPRESS 2D Fluorescence Scanner
- Perseptive BioCad Protein Fractionator
- Bio-Rad BioPlex multiplex cytokine analyzer
Mass Spectrometry Services: The Proteomics Core includes a Mass Spectrometry Facility. A wide range of analyses are available including MALDI-TOF, MALDI-TOF/TOF, electrospray (ESI) LC/MS and ESI-LC/MS/MS.
Services available are:
- Peptide mass fingerprint analysis
- Post-translational modification identification
- Stable isotope related procedures ( iTRAQ, and 18O and SILAC)
- Quality Control
- Proteomics Bioinformatics
Quality Control: Data quality is ensured through the use of appropriate internal and external standards. The MALDI-TOF mass spectrometers are calibrated with external standards that vary depending on the analyte molecular weight. This results in the best calibration for the most accurate mass assignments. Samples that have been trypsin digested are internally recalibrated from observed trypsin autolysis fragments. The electrospray mass spectrometers are calibrated using polyalanine which allows for a distribution of multiple mass peaks. A variety of search engines for protein identification from mass fingerprinting and for interpretation of MS/MS spectra are available each of which generates its own statistics. The choice of database searching software to be applied is optional to investigators and discussion of this matter is encouraged with Core personnel.
- Applied Biosystems Voyager-DE STR MALDI-TOF
- Waters/Micromass Qtof-2 with Waters Cap LC system
- Applied Biosystems 4700 Proteomics Analyzer (MALDI TOF/TOF)
- Applied Biosystems 4800 MALDI TOF/TOF Analyzer (COMING SOON)
- Applied Biosystems 4000 QTRAP with LC Packings capillary LC system
Proteomics Bioinformatics Services: The core utilizes Nonlinear Dynamics software, which quantifies stained protein images and calculates relative levels of expression in comparative studies. The software generates suggested spot picking lists as well as statistical analyses for estimating the significance of expression levels. After discussion with investigators, promising protein spots are then processed using robotic devices with identification accomplished utilizing the resources the Mass Spectrometry resources and database interrogation. In order to provide investigators with sample tracking, powerful data management capability, and facile data access, GenoLogics Proteus LIMS (Laboratory Information Management System) is also in place.
- The Protein Bioinformatics Core provides informatics tools, including statistical tools, for the analysis of 1D and 2D electrophoresis gels utilizing a software suite from Nonlinear Dynamics (Progenesis) and sample tracking, data management and access tools, using software from GenoLogics (Proteus LIMS). The BRF is a major Nonlinear and GenoLogics partner and collaborates with these manufacturers in the development of their software.
- The Bioinformatics Core interacts extensively with other BRF Cores, and UTMB Centers and Departments and has integrated the Progenesis software into high-end user groups. For example, the BRF has installed the Editor Progenesis software at a number of sites across the UTMB campus.
- Group training sessions are scheduled as needed, typically every six months.
- Analysis of gel images and quantification of protein expression utilizing Nonlinear Dynamics Progenesis Discovery Software.
- Validation of data utilizing statistical analysis
- Student's t-test: Hypothesis and significance testing
- Multiple Hypothesis testing corrections
- Hierarchical clustering: Control vs. Treated
- Analysis of Variance (ANOVA): Time course/dosage dependence of expression
- Nonlinear 1D gel analysis software suite
- Nonlinear Progenesis SameSpots with Statistical Package software suite for 2D gel analysis (16 floating licenses)
- Nonlinear Progenesis LC-MS for label-free LC-MS data analysis
- Genologics LIMS for data management and access
Molecular Genomics Core (MGC)
The mission of the MGC is to provide UTMB researchers access to cutting-edge technologies in functional genomics. These technologies are inherently expensive to acquire and operate, making it cost prohibitive for individual laboratories to invest in the necessary capital equipment. The MGC objective is to provide access not only to the technologies but also our experienced staff. In addition to the core platforms, we offer a number of services aimed at data validation. A central goal of the MGC is to provide high quality data in a timely fashion and excellent customer service in a collaborative spirit. The MGC currently provides analysis platforms for both genome (sequencing and genotyping) and transcriptome (gene expression) analysis. In addition to the ongoing application of existing technologies, the MGC is also actively engaged in the development and testing of new related technologies. The MGC is supported by Geneus, a custom Laboratory Information Management System (LIMS) that provides an easy-to-use secure interface for sample submission and data retrieval. Clients will access Geneus through LabLink, which allows a seamless integration of sample and data tracking with UTMB ITS Biorepository and proteomics cores. The MGC facilities will e available to OAIC investigators. They will be used by PES1.Molecular Genomics Core Website
- Real-Time PCR
- SNP genotype
- Nucleic acid isolation
- High Resolution Melt (HRM)
- Applied Biosystems 3130xl Genetic Analyzer
- Affymetrix Scanner
- GeneChip Fluidics Station 400
- Gene Pix4000B
- ABI Prism 7000 Sequence Detection System
- 7500 Real-Time PCR System
- Beckman Biomek FX
- NanoDrop® ND-1000 UV-Vis Spectrophotometer
- MJ Research Peltier Tetrad Thermal Cycler
- The Agilent 2100 Bioanalyzer
Next Generation Sequencing Core Facility (NGS)
UTMB’s Next Generation Sequencing (NGS) Core utilizes an Illumina HiSeq 1500 sequencing system to perform massively parallel sequencing for genetic analysis. The NGS Core offers support in library construction from various template sources; RNA (total, poly A+ and miRNA), chromatin immunoprecipitated DNA (ChIP-Seq) and DNA (genomic and amplicon-derived). Library complexity is assessed using qPCR prior to amplification. Illumina NGS technology uses adapter-ligated template molecules to populate a hollow glass flow cell. Individual target molecules are then amplified to create template clusters. Templates are sequenced using reversible, fluorescent-tagged terminator nucleotides. The HiSeq 1500 has the capacity to create over 1.5 billion clusters across each lane in an 8-channel flow cell. The 1500 HiSeq offers the versatility of using either an eight lane or a two lane flow cell. With the capability of either single-end or paired-end sequence reads and 50 to 100 base read lengths, the HiSeq 1500 can generate up to 300 Gb from a single sequence run. The two lane platform can also accommodate 150 base reads. Costs are reduced by indexing (“bar coding”) individual template libraries, allowing multiple libraries to be sequenced in each of the flow cell lanes. Investigators have the option of two-lane, eight-lane or first available in selecting the platform for their sequencing project.
What can you do with Next Generation Sequencing?
- De novo assembly
- Variant identification
- Transcriptome (reference-guided or de novo)
- miRNA and small ncRNA sequencing
- Quantitative - RNA-Seq expression analysis