Papers by Piriya Wongkongkathep
International Journal of Mass Spectrometry, 2015
The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined wit... more The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.
Molecular Cancer, 2015
Background: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has b... more Background: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes. Methods: To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed.
RATIONALERecent experiments utilizing photodissociation in linear ion traps have enabled signific... more RATIONALERecent experiments utilizing photodissociation in linear ion traps have enabled significant development of Radical-Directed Dissociation (RDD) for the examination of peptides and proteins. The increased mass accuracy and resolution available in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) should enable further progress in this area. Preliminary experiments with photoactivated radicals are reported herein.Recent experiments utilizing photodissociation in linear ion traps have enabled significant development of Radical-Directed Dissociation (RDD) for the examination of peptides and proteins. The increased mass accuracy and resolution available in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) should enable further progress in this area. Preliminary experiments with photoactivated radicals are reported herein.METHODSA 266 nm Nd:YAG laser is coupled to a FTICR or linear ion trap mass spectrometer. Radical peptides and proteins are generated by ultraviolet photodissociation (PD) and further activated by collisions or infrared photons.A 266 nm Nd:YAG laser is coupled to a FTICR or linear ion trap mass spectrometer. Radical peptides and proteins are generated by ultraviolet photodissociation (PD) and further activated by collisions or infrared photons.RESULTSA 266 nm UV laser and an IR laser can be simultaneously coupled to a 15 Tesla FTICR mass spectrometer. The ultra-low-pressure environment in FTICR-MS makes collisional cooling less competitive, and thus more secondary fragments are generated by UVPD than in linear ion traps. Activation by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) or infrared multiphoton dissociation (IRMPD) also yields additional secondary fragmentation relative to CID in an ion trap. Accurate identification of RDD fragments is possible in FTICR-MS.A 266 nm UV laser and an IR laser can be simultaneously coupled to a 15 Tesla FTICR mass spectrometer. The ultra-low-pressure environment in FTICR-MS makes collisional cooling less competitive, and thus more secondary fragments are generated by UVPD than in linear ion traps. Activation by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) or infrared multiphoton dissociation (IRMPD) also yields additional secondary fragmentation relative to CID in an ion trap. Accurate identification of RDD fragments is possible in FTICR-MS.CONCLUSIONSRelative to linear ion trap instruments, PD experiments in FTICR-MS are more difficult to execute due to poor ion cloud overlap and the low pressure environment. However, the results can be more easily interpreted due to the increased resolution and mass accuracy. Copyright © 2014 John Wiley & Sons, Ltd.Relative to linear ion trap instruments, PD experiments in FTICR-MS are more difficult to execute due to poor ion cloud overlap and the low pressure environment. However, the results can be more easily interpreted due to the increased resolution and mass accuracy. Copyright © 2014 John Wiley & Sons, Ltd.
“Native” mass spectrometry (MS) has been proven to be increasingly useful for structural biology ... more “Native” mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains. Figure ᅟ
Developmental cell, 2013
The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small tr... more The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.
The Journal of biological chemistry, 2014
The molecular tweezer, CLR01, binds to Lys and prevents aggregation of ␣-synuclein. Results: CLR0... more The molecular tweezer, CLR01, binds to Lys and prevents aggregation of ␣-synuclein. Results: CLR01 binds directly to monomeric ␣-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure.
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Papers by Piriya Wongkongkathep