Proteomics, Metabolomics, Metabolic pathway analysis

My current research is focused on the molecular mechanism study and biomarker discovery of metabolic diseases, in particular non-alcoholic fatty liver disease (NAFLD), using high-resolution and high-throughput mass spectrometry-based proteomics, lipidomics, metabolomics and 13C-MFA approaches in combination with physiological assessments and biochemical assays. We develop proteomics, lipidomics, metabolomics and 13C-MFA methods in this context and use the methods in other collaborative projects.

I am recruiting energetic, creative and highly motivated Master’s and PhD students (Those with experience in mass spectrometry-based proteomics/metabolomics, bioinformatics, 13C-MFA, cell culture are beneficial but not absolutely required) in the following arears but not limited to:

1. Role of Ferroptosis in Non-alcoholic Fatty Liver Disease (NAFLD): NAFLD involves accumulation of fat in the liver and can progress to non-alcoholic steatohepatitis (NASH), a leading cause of liver disease-related mortalities. Both NAFLD and NASH are risk factors for contracting type 2 diabetes and cardiovascular disease. The prevalence of NAFLD is closely associated with obesity, with 90% of the obese population afflicted with NAFLD. Currently there is no effective drug treatment for NAFLD and NASH. The reason for this is that we do not fully understand the mechanisms underlying NAFLD progression. We are testing the hypothesis that ferroptosis, a new type of regulated cell death, which is initiated by severe lipid peroxidation caused by reactive oxygen species (ROS) generated from the oxidation of excessive fats in NAFLD, plays a critical role in the progression to NASH. Lipid peroxidation directly causes the damage of the membrane of cells and organelles, importantly the products of lipid peroxidation are reactive and modify proteins, nucleic acids and other lipids, impairing the cell’s detoxification function and serves as signal molecules driving cell death and inflammation response. Our research focuses on the quantitative identification of the toxic oxidized lipid species in organelles and the contribution of the oxidized organelles to ferroptosis sensitivity, proteomics, lipidomics and 13C-MFA to elucidate the key pathways that regulate and lead to cell death and inflammation (onset of NASH). Human liver primary or cell lines (e.g., HepG2 cells) and organoid cells (e.g., HepRG) will be used as the model systems.

2. Proteomics: We develop mass spectrometric strategies to identify and characterize peptides and proteins in complex biological materials, including intact cells, whole organisms, tissues, biofluids, organelles, purified proteins or protein complexes and biological preparations such as vaccines. Our lab is equipped with a state-of-the-art ThermoFisher Orbitrap Fusion Lumos Tribrid ETD mass spectrometer, fronted by a nanoflow UPLC (Ultimate 3000) and a high-flow UPLC (Vanquish). The ultra-high scan speed and resolution, superior sensitivity and mass accuracy, ETD/EThcD capable instrument enables a large number of different experiments to be implemented and makes detection of very low abundance proteins and lipids easy, fast and reproducible. The lab also has an ABSCIEX MALDI-TOF/TOF mass spectrometer with mass spectrometry imaging (MSI) capability that provides fast and confident identification of proteins and MSI on tissue sections. We develop workflows and methods through our own research themes and through collaborations with researchers within the University and from outside institutions in a wide range of projects for:

• Protein identification in complex biological samples from cells, organisms, tissues, biofluids, subcellular organellar fractions (e.g., microsomes, secretomes, exosomes, and the surfaceome), gel bands, biological preparations such as vaccines, and recombinant proteins. We are particularly interested in developing strategies for enrichment and identification of membrane and low abundance proteins.
• Protein quantitation assessing differential protein expression under different conditions by label-free and/or isobaric tandem mass labelled (e.g., TMT) quantitation approaches. The TMT labelling enables the simultaneous analysis of 16 samples (16 Plex TMT reagent) on the Fusion Lumos platform that enables high throughput fast and confident biomarker discovery in a large number of samples such as clinical samples.
• Post-translational modification (PTM) characterisation such as glycosylation, phosphorylation, and ubiquitination of proteins and peptides can be achieved using the Lumos mass spectrometer with high coverage.
• Intact protein and natural peptide analysis (Top-down proteomics), including determination of molecular mass, characterisation of any post-translational modifications, protein isoforms, and protein sequences through fragmentation of the intact proteins and peptides using ETD/ETchD within the Fusion Lumos mass spectrometer.
• Protein-protein interactions and protein complexes using the latest mass spectrometry-compatible cross-linking methods and the Fusion Lumos mass spectrometer platform.
• Data independent acquisition (DIA) for superior quantification accuracy, precision and reproducibility by continuous collection of fragment-ion spectra over a preselected precursor range.
• Mass spectrometry imaging (MSI) using MALDI imaging that maps the spatial distribution of tens to hundreds of proteins, peptides, lipids, metabolites or drugs in thin tissue sections. Using this technique, it is possible to map metabolic interactions between tissues in a variety of physiological conditions.

3. Lipidomics and Metabolomics: We are establishing lipidomics and chemical analysis capabilities for comprehensive identification and quantification of diverse metabolites over a wide range of concentrations. We are developing and employing a variety of analytical biochemistry techniques, including novel sample extraction and ultra-high resolution tandem mass spectrometry (MS/MS) such as HCD MS and CID MS2/MS3 that can only be achieved on the Fusion Tribrid instrument fronted with a high-flow UPLC (Vanquish). We are using these techniques to detect changes associated with dysregulation of normal cellular function in complex lipids and other metabolites abundances and determine their molecular formulae and structure. Software packages available include FreeStyle, CompoundDiscoverer, LipidSearch, and TraceFinder.

4. 13C-Metabolic Flux Analysis: This involves applying stable isotope tracers, e.g., 13C, and analysing the mass isotopomer distributions (MIDs) of the intracellular metabolites using mass spectrometry and quantifying the metabolic fluxes in complex metabolic pathways of the cell. The MIDs provide direct information about the metabolic activities of pathways associated with a specific metabolite (a node) and allow global quantification of the entire metabolic network. Therefore, 13C-MFA can identify important regulatory nodes of the metabolic network in pathological conditions or in response to different stimuli. I am interested in developing LCMS methods for measurement of MIDs using the Vanquish Lumos system and 13C-MFA computation using online available software by in-house scripting.

5. Bioinformatics for proteomics and metabolomics: I am collaborating with Professor Mengjie Zhang in evolution computation in School of Computer Science and Engineering at the University in developing algorithms and tools for mass spectrometry-based proteomics and metabolomics data processing and interpretation.

Contact Dr. Lifeng Peng (lifeng.peng@vuw.ac.nz) if you are interested in doing a PhD or a Master’s or a collaborative research in the above areas.