- Library Home /
- Search Collections /
- Open Collections /
- Browse Collections /
- UBC Theses and Dissertations /
- Comparison of formaldehyde with established crosslinkers...
Open Collections
UBC Theses and Dissertations
UBC Theses and Dissertations
Comparison of formaldehyde with established crosslinkers to characterize transient protein structures using mass spectrometry Srinivasa, Savita
Abstract
Chemical cross-linking along with mass spectrometry can elucidate protein geometry by introducing stabilizing covalent linkages as distance constraints. Formaldehyde’s small size allows it to quickly permeate the cellular membrane without external manipulation and preserve close-proximity and transient protein interactions under physiological conditions. Despite its established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied to structural proteomics, which other cross-linkers have already accomplished. In this thesis, formaldehyde along with four other established cross-linkers (three Nhydroxysuccinimide ester cross-linkers and one zero-length cross-linker), varying in size and reactivity, were shown to capture Ribonuclease S and the Ca²⁺-free calmodulin-melittin, which are two weak protein complexes. It was demonstrated that the yield of close-proximity crosslinking from zero-length and formaldehyde cross-linkers reflected the dissociation constants of both transient complexes. A comparison between the identification of formaldehyde and established crosslinked species via first stage mass spectrometry (MS) and tandem mass spectrometry (MS/MS) provided insight into what evidence is sufficient to confirm formaldehyde cross-linked species. Cross-linked species from all cross-linkers were identified via MS/MS in the Ca²⁺-free calmodulin-melittin. These were used to impose different distance constraints to examine the unknown binding orientation of Ca²⁺-free calmodulin to melittin. The relatively straightforward discovery of N-hydroxysuccinimide ester cross-linking was offset by its large size and ambiguous distance constraints that may not be suitable for small proteins. Although zero-length cross-linkers create close proximity linkages, the high abundance of its reactive sites in calmodulin-melittin produced diversified products, complicating mass spectrometric detection. The increased complexity in identifying formaldehyde reaction products via mass spectrometry was due to its reactivity with several amino acids. This work represents the first report of formaldehyde cross-links identified between non-covalently associated protein components, supporting formaldehyde’s ability to stabilize weak interactions. Four formaldehyde crosslinking sites were localized in calmodulin-melittin, and the mechanisms of the formation of these cross-links were revealed using in vivo-like conditions. The uniformity of formaldehyde crosslink localization reflected the uniform binding structure of calmodulin. Furthermore, the binding orientation of calmodulin and melittin captured by formaldehyde was shown to be most consistent with recent literature compared to the other cross-linkers.
Item Metadata
Title |
Comparison of formaldehyde with established crosslinkers to characterize transient protein structures using mass spectrometry
|
Creator | |
Publisher |
University of British Columbia
|
Date Issued |
2017
|
Description |
Chemical cross-linking along with mass spectrometry can elucidate protein geometry
by introducing stabilizing covalent linkages as distance constraints. Formaldehyde’s
small size allows it to quickly permeate the cellular membrane without
external manipulation and preserve close-proximity and transient protein interactions
under physiological conditions. Despite its established uses in biology and
compatibility with mass spectrometry, formaldehyde has not yet been applied to
structural proteomics, which other cross-linkers have already accomplished. In
this thesis, formaldehyde along with four other established cross-linkers (three Nhydroxysuccinimide ester cross-linkers and one zero-length cross-linker), varying
in size and reactivity, were shown to capture Ribonuclease S and the Ca²⁺-free
calmodulin-melittin, which are two weak protein complexes. It was demonstrated
that the yield of close-proximity crosslinking from zero-length and formaldehyde
cross-linkers reflected the dissociation constants of both transient complexes. A
comparison between the identification of formaldehyde and established crosslinked
species via first stage mass spectrometry (MS) and tandem mass spectrometry
(MS/MS) provided insight into what evidence is sufficient to confirm
formaldehyde cross-linked species. Cross-linked species from all cross-linkers
were identified via MS/MS in the Ca²⁺-free calmodulin-melittin. These were used
to impose different distance constraints to examine the unknown binding orientation
of Ca²⁺-free calmodulin to melittin. The relatively straightforward discovery
of N-hydroxysuccinimide ester cross-linking was offset by its large size and
ambiguous distance constraints that may not be suitable for small proteins. Although
zero-length cross-linkers create close proximity linkages, the high abundance
of its reactive sites in calmodulin-melittin produced diversified products,
complicating mass spectrometric detection. The increased complexity in identifying
formaldehyde reaction products via mass spectrometry was due to its reactivity
with several amino acids. This work represents the first report of formaldehyde
cross-links identified between non-covalently associated protein components, supporting formaldehyde’s ability to stabilize weak interactions. Four formaldehyde
crosslinking sites were localized in calmodulin-melittin, and the mechanisms of
the formation of these cross-links were revealed using in vivo-like conditions. The
uniformity of formaldehyde crosslink localization reflected the uniform binding
structure of calmodulin. Furthermore, the binding orientation of calmodulin and
melittin captured by formaldehyde was shown to be most consistent with recent
literature compared to the other cross-linkers.
|
Genre | |
Type | |
Language |
eng
|
Date Available |
2017-06-21
|
Provider |
Vancouver : University of British Columbia Library
|
Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
|
DOI |
10.14288/1.0348399
|
URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
|
Graduation Date |
2017-09
|
Campus | |
Scholarly Level |
Graduate
|
Rights URI | |
Aggregated Source Repository |
DSpace
|
Item Media
Item Citations and Data
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International