Development of chromogenic cross-linkers and selective gas-phase dissociation methods to assess protein macromolecular structures by mass spectrometry
| dc.contributor.advisor | Brodbelt, Jennifer S. | en |
| dc.contributor.committeeMember | Marcotte, Edward M. | en |
| dc.contributor.committeeMember | Shear, Jason B. | en |
| dc.contributor.committeeMember | Stevens, Scott W. | en |
| dc.contributor.committeeMember | Stevenson, Keith J. | en |
| dc.creator | Gardner, Myles Winston | en |
| dc.date.accessioned | 2010-08-05T16:19:46Z | en |
| dc.date.accessioned | 2010-08-05T16:20:02Z | en |
| dc.date.accessioned | 2017-05-11T22:20:02Z | |
| dc.date.available | 2010-08-05T16:19:46Z | en |
| dc.date.available | 2010-08-05T16:20:02Z | en |
| dc.date.available | 2017-05-11T22:20:02Z | |
| dc.date.issued | 2009-12 | en |
| dc.date.submitted | December 2009 | en |
| dc.date.updated | 2010-08-05T16:20:02Z | en |
| dc.description | text | en |
| dc.description.abstract | Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.uri | http://hdl.handle.net/2152/ETD-UT-2009-12-513 | en |
| dc.language.iso | eng | en |
| dc.subject | Mass spectrometry | en |
| dc.subject | Infrared multiphoton dissociation | en |
| dc.subject | Cross-linking | en |
| dc.subject | Quadrupole ion trap | en |
| dc.subject | Photodissociation | en |
| dc.subject | Protein cross-linking | en |
| dc.subject | Electron transfer dissociation | en |
| dc.subject | Ultraviolet photodissociation | en |
| dc.subject | Small-interfering RNA | en |
| dc.title | Development of chromogenic cross-linkers and selective gas-phase dissociation methods to assess protein macromolecular structures by mass spectrometry | en |
| dc.type.genre | thesis | en |