Plant Metabolite Imaging
We are developing mass spectrometric imaging techniques to map metabolite distributions within plant tissues, and eventually among individual plant cells. Mass spectrometry not only allows positive identification of the many metabolites but can also reveal the substrates and precursors involved in each metabolic pathway. Such information will provide unprecedented details on the distribution of metabolites from cell to cell, cooperative and antagonistic effects among the metabolites, and environmental influences on metabolism. Such details will ultimately lead to a predictive understanding of the mechanisms that multicellular organisms use to regulate metabolic processes.
We are specifically focused on the model plant Arabidopsis. By studying the diversity of various metabolites and their locations, we hope to gain detailed insight into their biosynthesis as a function of genetics, tissue type, development, and environment. In analogy to matrix-assisted laser desorption ionization (MALDI), a laser beam will be used to interrogate sequential micrometer-sized areas of a plant by desorbing and ionizing the surface contents of the tissue into a mass analyzer. Rastering of the laser beam over the tissue will produce a laterally resolved-image of the various substances within different structures of the plant. Repeated vaporization at the same focused point of a plant structure will produce a depth profile of the components. We plan to generate ions directly from the plant tissue by designing novel additives as pseudo-matrixes. By minimizing sample preparation, compositional integrity and spatial resolution of the analysis will be guaranteed.
We use high mass resolution MALDI-LTQ-Orbitrap for this research and recently achieved a high spatial resolution (down to ~12mm). We are further developing the technique to achieve higher spatial resolution and find best application of this top notch technology. Recently we systematically studied various plant tissues of Arabidopsis cer1 mutant compared with wild type, and observed interesting biological phenomena with very detailed spatial resolution and high mass resolution.
This project is being performed in close collaboration with Basil J. Nikolau, R. Sam Houk, and Edward S. Yeung.
- Sangwon Cha, Zhihong Song, Basil J. Nikolau, and Edward S. Yeung, "Direct Profiling and Imaging of Epicuticular Waxes on Arabidopsis thaliana by Laser Desorption/Ionization Mass Spectrometry Using Silver Colloid as a Matrix", Anal. Chem. 2009, 81, 2991â€“3000.
- Sangwon Cha, Hui Zhang, Hilal I. Ilarslan, Eve Syrkin Wurtele, Libuse Brachova, Basil J. Nikolau and Edward S. Yeung, â€œDirect profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption ionization mass spectrometryâ€, The Plant Journal (2008) 55, 348â€“360.
- Sangwon Cha and Edward S. Yeung, â€œColloidal Graphite-Assisted Laser Desorption/Ionization Mass Spectrometry and MSn of Small Molecules. 1. Imaging of Cerebrosides Directly from Rat Brain Tissueâ€, Anal. Chem. 2007, 79, 2373-2385.
- Hui Zhang, Sangwon Cha, and Edward S. Yeung, â€œColloidal Graphite-Assisted Laser Desorption/Ionization MS and MSn of Small Molecules. 2. Direct Profiling and MS Imaging of Small Metabolites from Fruitsâ€, Anal. Chem. 2007, 79, 6575-6584.
Cross-linking Mass Spectroscopy
Determination of the 3D structure of all proteins, aimed by Structural Genomics, seems to be an unachievable goal with the current x-ray crystallography or NMR technologies only as signified by a low success rate of 3-5% to obtain genome-wide 3D protein structures (Yee et al., 2003). Moreover, the structural proteomics of biological complexes is expected to be a far more daunting process (Sall, 2003). The chemical cross-linking of intact proteins or protein complexes followed by enzymatic digestion and mass spectrometric analysis has been suggested as a low-resolution alternative (Sinz, 2006; Lee, 2008). This technique has been successfully utilized to provide protein structures and protein-protein interactions. It has also been utilized to construct high resolution protein structure by combining with x-ray crystallography of N- and C-terminal domains (Forwood et al., 2007). However, inherent difficulty in finding low abundance cross-linked peptides out of thousands of other coexisting non-cross-linked peptides has been a bottleneck in its further development (Sinz, 2006).
We developed a high throughput and high sensitivity shotgun approach to efficiently identify cross-linked peptides (Lee et al., 2007). Our new method is arguably the most sensitive than any other approach reported so far and we further improved this approach by adopting probability based scoring system to significantly remove false positives (Lee, 2009). In the database search against large protein sequences, we found partial matching could be a significant problem in cross-linking sites search and we suggested a novel approach to remove such inadvertent false positives by adopting E-value filtering in each peptide level (See Figure below). It is our expectation that this approach has great potential to advance cross-linking mass spectrometry as a unique tool supplementary to x-ray crystallography or NMR. Our long term goal is to advance cross-linking mass spectrometry to fill the current technological gap in structural biology. We currently have several collaborations with biologists, computational scientists, and biophysicists to solve the structural proteomics problem together.
- Adelinda Yee, Keith Pardee, Dinesh Christendat, Alexei Savchenko, Aled M. Edwards, and Cheryl H. Arrowsmith "Structural Proteomics: Toward High-Throughput Structural Biology as a Tool in Functional Genomics", Acc. Chem. Res. 36, 183-189, 2003.
- Andrj Sall, "NIH Workshop on Structural Proteomics of Biological Complexes", Structure, 11, 1043-1047, 2003.
- A. Sinz, â€œChemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactionsâ€, Mass Spectrom. Rev. 25, 663-682, 2006.
- Young Jin Lee, Christopher B. Whitehurst, Erik J. Soderblom, Michael B. Goshe, Dennis T. Brown, Brett S. Phinney, â€œCross-linking Site Mapping of Sindbis Virus Structural Proteinsâ€, Proceed. 54th ASMS Conference on Mass Spectrom. Allied Topics, MP546, 2006.
- Young Jin Lee, Robert H. Rice, and Young Moo Lee, â€œProteome Analysis of Human Hair by Two Dimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry (2-D LC-MS/MS): From Protein Identification to Posttranslational Modificationâ€, Mol. Cell. Proteomics, 5, 789-800, 2006b.
- Young Jin Lee, Laura Lachner, Jodi Nunnari, Brett S. Phinney, â€œShotgun Cross-linking Analysis for Studying Quaternary and Tertiary Protein Structuresâ€, J. Proteom Res. 6, 3908-3917, 2007.
- Young Jin Lee, â€œMass Spectrometry based Cross-linking Sites Mapping for Structural Elucidation of Protein and Protein Complexâ€, Molecular Biosystems, 4, 816, 2008.
Young Jin Lee, â€œProbability based Shotgun Cross-Linking Sites Analysisâ€, J. Am. Soc. Mass Spectrom., in print, accepted on Jun 30th, 2009.
- J. K. Forwood, A. S. Thakur, G. Guncar, M. Marfori, D. Mouradov, W. Meng, J. Robinson, T. Huber, S. Kellie, J. L. Martin, D. A. Hume and B. Kobe, â€œStructural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammationâ€, Proc. Natl. Acad. Sci. U. S. A., 104, 10382â€“10387, 2007.
The interest in renewable energy is getting great attention ever, to have our nation to be energy independent and achieve affordable gas price. Among the renewable energies, bio-oil (or bio-fuel) is especially important as an alternative transportation fuel. Recent corn based bio-ethanol production invoked many issues such as high corn price and food affordability to human and/or animals. As analytical chemists, we are contributing to this research field by developing and providing analytical tools. We have two research projects regarding the bio-oil analysis of non-food stock bio-oils.
Algae Oil Sequestration (in Collaboration with Victor Lin)
Algae has a great potential as a source of renewable energy (taken from oilgae.com below)
* The yields of oil and fuels from algae are much higher (10-100 times) than competing energy crops.
* Algae can grow practically anywhere, thus ensuring that there is no competition with food crops.
* Algae are excellent bioremediation agents - they have the potential to absorb massive amounts of CO2 and can play an important role in sewage and wastewater treatment.
* Algae are the only feedstock that have the potential to completely replace world's consumption of transportation fuels.
However, its development as a renewable energy source currently has many limitations. Among those is how to efficiently collect and separate diesel-like oil components from algae. MSN (mesoporous silica nanoparticle) or MCN (mesoporous carbon nanoparticle) developed by Victor Lin may solve this problem from its size selective affinity with a certain size of fatty acids. We are developing mass spectrometry based analytical platform to monitor algae bio-oils in collaboration with Victor Linâ€™s group.
Petroleomic Analysis of Fast Pyrolysis Bio-oil
Another promising research field in bio-oil is fast pyrolysis of bio-masses. Dr. Robert C. Brown in Mechanical and Chemical Engineering is a prominent engineer in this research field. However, the lack in molecular understanding of thus generated bio-oils has been a critical bottleneck for the further development of the technology and optimization of the involved chemical processes. The analysis of fast pyrolysis bio-oils has been limited to volatile compound analysis such as GC-MS while non-volatile compounds are more important as bio-diesel. We recently developed a petroleomic approach to analyze hundreds of compounds in bio-oils using high resolution mass spectrometry. It is the first time comprehensive analysis of this type of bio-oils and we could hypothesize the fast pyrolysis breaks liginin polymers down to dimer units.