Dissociation methods for glycans
The MSR is developing activated electron dissociation (ExD) methods for glycans. The goal of these projects is to maximize the ability to sequence glycans in a single stage of tandem MS, compatible with on-line LC-MS. This entails investigation of ExD mechanisms and development of data analysis software.
Electron Capture Dissociation (ECD) was first applied to biomolecules by Zubarev et al. (1) from the McLafferty Group. ECD has the remarkable and useful ability to produce odd-electron, free-radical driven fragmentation of the same type as are generated by EI mass spectrometry (2). This fragmentation method generates different and often complementary fragmentation patterns when compared with Collisionally Activated Dissociation (CAD), Infrared Multiphoton Dissociation (IRMPD), or other slow, even-electron fragmentation methods.
For proteins, ECD cleaves between the backbone amide and the alpha carbon to form C and Z* fragments (where “*” refers to the radical “dot” symbol which unfortunately is unavailable on this keyboard). Moreover, the cleavage sites show very little selectivity for particular amino acids with the two exceptions of disulphide bonds (having high radical affinity) and proline (which is cyclic around the amide – alpha carbon and therefore requires breaking 2 bonds).
Additionally, for proteins, labile post-translational modifications such as phosphorylation sites, o-glycosylation sites, n-glycosylation sites, and others remain attached to the backbone during ECD MS/MS experiments allowing determination of the site and identity of post-translational modifications.
We are currently investigating the uses of ExD for the analysis of carbohydrates and glycoconjugates
Here are some publications from this project:
- Yu, X.; Huang, Y.; Lin, C.; Costello, C. E. Energy-dependent electron activated dissociation of metal-adducted permethylated oligosaccharides. Anal. Chem. 2012, 84, 7487-94. Pubmed Link
- Yu, X.; Jiang, Y.; Chen, Y.; Huang, Y.; Costello, C. E.; Lin, C. Detailed glycan structural characterization by electronic excitation dissociation. Anal. Chem. 2013, 85, 10017-21. Pubmed Link
- Huang, Y.; Yu, X.; Mao, Y.; Costello, C. E.; Zaia, J.; Lin, C. De Novo Sequencing of Heparan Sulfate Oligosaccharides by Electron-Activated Dissociation. Anal. Chem. 2013, 85, 11979-11986. Pubmed Link
- Hu, H.; Huang, Y.; Mao, Y.; Yu, X.; Xu, Y.; Liu, J.; Zong, C.; Boons, G. J.; Lin, C.; Xia, Y.; Zaia, J. A Computational Framework for Heparan Sulfate Sequencing Using High-resolution Tandem Mass Spectra. Molecular & cellular proteomics : MCP 2014, 13, 2490-2502. Pubmed Link
- Huang, Y.; Pu, Y.; Yu, X.; Costello, C. E.; Lin, C. Mechanistic Study on Electronic Excitation Dissociation of the Cellobiose-Na(+) Complex. J Am Soc Mass Spectrom 2016, 27, 319-28. Pubmed Link
- Hong, P.; Sun, H.; Sha, L.; Pu, Y.; Khatri, K.; Yu, X.; Tang, Y.; Lin, C. GlycoDeNovo – an Efficient Algorithm for Accurate de novo Glycan Topology Reconstruction from Tandem Mass Spectra. J Am Soc Mass Spectrom 2017, 28, 2288-2301. Pubmed Link