2015.09.14 10:56
9월10일(목)/Dr. Lijljana Pasa-Tolic(PNNL, USA)/Towards high-throughput top-down mass spectrometry
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| 초청강사 | Dr. Lijljana Pasa-Tolic |
|---|---|
| 소속 | PNNL, USA |
| 일시 | 2015년 9월 10일(목) 오후 5시 |
| 장소 | 아산이학관 331호 |
Towards high-throughput top-down mass spectrometry
Abstract:
Combinatorial post-translational modifications (PTMs), signal peptide cleavages, proteolytic processing and
site mutations are all important biological processes that largely go undetected in traditional bottom-up
proteomic analyses. While several PTMs are successfully identified using bottom-up methods, information
including stoichiometry of modifications on a single proteoform, [Smith LM et al. Nat Methods. 2013, 10
(3):186-7] or presence of a combination of multiple modifications on a single proteoform, is practically
impossible to infer from peptide-level data. Hence, top-down (i.e. intact protein) mass spectrometry (MS)
enables physiologically relevant studies of microbes and higher eukaryotes and are rapidly becoming an
important avenue for proteomic studies.
Recent advances in MS instrumentation, separation, and bioinformatics significantly increased the throughput,
comprehensiveness and sensitivity of top-down MS for proteomic applications. Selected examples from our lab
include microbial proteomics; broad characterization of modifications on intact Salmonella proteins
potentially relevant to pathogen biology; characterization of the native forms of human salivary proteins
potentially relevant to oral salivary diagnostics; and insights into the underexplored mechanism of epigenetic
control of gene expression for e.g. generating profitable bioactive compounds in fungus.
There are, however, considerably more technical challenges associated with nearly all aspects of the top-down
approach. Perhaps the biggest challenge relates to MS performance. Fourier transform ion cyclotron resonance
(FTICR) offers the highest mass resolving power and accuracy of any mass analyzer. And, because all key
measures of FTICR MS performance improve with increased magnetic field strength, a high-field FTICR
spectrometer arguably provides that next level of performance needed to bring the top-down MS to the
mainstream. Initial results acquired using a 21T FTICR spectrometer, recently brought online at EMSL, a
national scientific user facility operated by PNNL for the U.S. Department of Energy (DOE), will be presented.
The spectrometer also incorporates advanced tandem MS capabilities (including UV photodissociation) and will
enable efficient characterization of proteins 2-4 times larger than presently attainable, thus facilitating
the characterization of the functional proteomes.