Browsing by Author "Burns, Kyle"

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    Advances in Hydrogen Exchange Mass Spectrometry to Study Microtubules and MAPs
    (2015-05-27) Burns, Kyle; Schriemer, David
    Microtubules are a fundamental component to the cellular cytoskeleton and are responsible for cell structure, motility, intracellular transport, and formation of the mitotic spindle. The ability to serve the diverse functions requires constant flux between polymerization and depolymerization. The regulation of microtubule dynamics is accomplished by microtubule associated proteins that can interact with microtubules, or its tubulin dimer, to induce polymerization or depolymerization. The depolymerization mechanism induced by mitotic centromere-associated kinesin (MCAK) was studied in detail. Studying a large protein system such as the regulation of microtubule dynamics at the molecular level requires an integrative structural biology approach. Hydrogen exchange mass spectrometry (HX-MS) is a vital technique for studying the protein dynamics and was optimized for the analysis of large protein complexes. Two HX-MS platforms consisting of a FT-MS and a high-resolution QTOF mass spectrometer were evaluated by comparing the figures-of-merit for a typical bottom-up HX-MS experiment: peptide identification, deuterium measurement accuracy, and deuterium measurement precision. The Orbitrap Velos identified 64% more peptides than the TripleTOF 5600, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the TripleTOF, depending on the Orbitrap resolution setting; however, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate. The findings presented support the use of the TripleTOF 5600 for further development of hydrogen exchange methods. A data-independent acquisition approach was developed that combines peptide fragmentation data and a new peptide scoring algorithm (WUF, Weighted Unique Fragment) to provide MS/MS data for HX measurements while reducing manual validation. The scoring incorporates elements of the validation process and preserves high peptide identification accuracy. When compared to a conventional Mascot-driven HX-MS method, HX-MS2 produces two-fold higher tubulin sequence depth at a peptide utilization rate of 74%. The HX-MS2 method was applied to study the microtubule depolymerization process induced by MCAK. In the described model, the N terminus is responsible for the lateral separation in conjunction with the outward curvature induced by the motor domain. The C terminus is responsible for regulating the microtubule interactions.