The Proteogenomics Core comprises a group of experts with experience in the implementation of the wide range of approaches in molecular biology, experimental design, and downstream computational analyses. Core C has a well established track record of innovating novel and cutting-edge methodologies in both next-generation sequencing and mass spectrometry, and can therefore develop and provide state-of-the-art technical support to advance the proposed research to new frontiers. The overarching goal of the Proteogenomics Core is to provide a comprehensive and unprecedented characterization of the “phenome” of cells infected by EBOV, both in cell culture and pre-clinical models.
Haiping Hao
For this project, Dr. Hao will be involved in planning the sequencing strategies, performing quality control on the results, and performing preliminary analyses on raw data for Core C.
Hao Lab – Molecular Genetics Facility (MGF): Next Generation Sequencing Core:
Website: Hao Lab – Molecular Genetics Facility (MGF): Next Generation Sequencing Core: website
Lab Phone: (409) 772-6349
William Russell
Dr. Russell will design and direct proteomic experiments for identifying and quantifying peptides/proteins using label-free quantitation as part of Core C. He has access to equipment in the UTMB Mass Spectrometry Facility, including a ThermoScientific Orbitrap Fusion mass spectrometer, a Sciex 5600 QTOF System, a 6500 Qtrap LC-MS/MS System, and a 5800 MALDI TOF/TOF System. A Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer was added to the facility recently (2020). All instrumentation is equipped with the necessary liquid chromatography (Dionex 3000 nRSLC system or Agilent 1260 system) and data analysis programs. In addition, the facility has an extremely capable staff skilled in proteomic and posttranslational modification analysis, including phosphorylation, palmitoylation, SUMOlytion, and acetylation.
Russell Lab – UTMB Mass Spectrometry Core Facility (MSF)
Website: Russell Lab website
Lab Phone: (409)772-6338
We specialize in 3 main sub-fields of LC-MS-based bioanalytical chemistry:
- Large scale proteomic analyses
- Sample prep workflows compatible with deactivated infectious agents
- The PO1 was chosen to use DIA as a quantitative proteomic strategies, although others are available (TMT (isobaric label), DDA Label-free)
- Differential quantitative analysis of post-translational modifications (PTM’s)
- State-of-the-art proteomic analysis capabilities include nano-flow UHPLC coupled to Orbitrap Eclipse with FAIMS-Pro differential ion mobility (LC-FAIMS).
- Multiple offline HPLC-UV-based fractionation options for complex protein mixtures available
- Purified protein primary structure characterization
- High resolution intact mass analysis in denaturing and native conditions
- High resolution peptide mapping (sequence confirmation, PTM localization)
- Large scale (targeted) Lipidomic / Metabolomic analyses
Proposed methods for protein and PTM analysis of Ebola-infected cells, (see image below):
- Human primary cells will be lysed in 5% SDS, thermally denatured, and protein fractions will be recovered.
- Protein samples will be individually prepared, and trypsin digested.
- Enrichment of ubiquitin modified peptides will be accomplished using antibodies directed against the C-terminal di-glycine-remnant of ubiquitin which is conjugated at lysine sites of ubiquitinated proteins.
- Antibody-bound peptides and unbound fractions from individual samples will be labeled using the Tandem Mass Tag (TMT) isobaric labeling technology.
- Antibody-bound fractions from each sample will be eluted, combined and analyzed LC-FAIMS.
- Anti-GG-peptide unbound fractions from each sample will be separately TMT-labeled then combined and enriched for phosphorylation using immobilized metal affinity chromatography (IMAC).
- Recovered phosphorylated peptides will be analyzed by LC-FAIMS.
- Unbound proteome (flow through from step 7) will be further fractionated using high pH reversed phase and each fraction will be separately analyzed by LC-FAIMS.
- Data acquired from steps 5, 8, and 9 will be searched and resulting PTM and peptide information sent to the modeling core.