Glycopeptide Enrichment Workflows for Downstream Mass Spectrometric Analysis
| dc.contributor.author | Bodnar, Edward | |
| dc.contributor.examiningcommittee | O'Neil, Joe (Chemistry) Krokhin, Oleg (Internal Medicine) Butler, Mike (Microbiology) Li, Liang (University of Alberta, Chemistry) | en_US |
| dc.contributor.supervisor | Perreault, Helene (Chemistry) | en_US |
| dc.date.accessioned | 2015-07-30T23:14:41Z | |
| dc.date.available | 2015-07-30T23:14:41Z | |
| dc.date.issued | 2013-11-01 | en_US |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | Mass spectrometry (MS) is a power analytical tool which is capable of analyzing biomolecules in great detail, both structurally and quantitatively. With regards to glycans, special considerations regarding sample preparation are necessary in order to achieve reproducible identification and relative quantification of these analytes. A workflow for isolation at the glycopeptide level and subsequent detection at the glycan level with phenylhydrazine, demonstrated that monoclonal antibodies (mAbs) containing a specific amino acid mutation were able to express approximately an additional 50% of the α2,6 disialylated glycan compared to their non-mutant analogues. In a second experiment using mAbs, an azide modified glycan (Ac4ManAz) was introduced both metabolically and enzymatically during mAb production. This glycan is a precursor in the sialic acid pathway and the azide moiety allows for specific chemistry post-production including the potential for highly specific enrichment. The results of this workflow demonstrated that [100 μM] of Ac4ManAz precursor added to the cell media was necessary for metabolic expression. More complex samples however, may contain multiple sites of glycosylation. To conserve the site of attachment, these molecules are often studied at the glycopeptide level, and require enrichment of glycopeptides to improve the lower signal intensity observed in the presence of co-eluting peptides. Carboxymethyl chitosan (CMCH) as well as amine-functionalized magnetic-nanoparticles (MNP) were developed as novel materials for this purpose. CMCH is naturally occurring, and therefore is cost-effective and readily available. In a 12 protein mixture CMCH demonstrated the bulk enrichment of glycopeptides yielding an approximately 20% higher enrichment of sialylated species as compared to a commercially available glycopeptide kit through the use of tandem mass tags for relative quantification. In the same approach, amine functionalized MNP were produced and used to enrich glycopeptides from tryptic digests. This approach was fast (about 10 mins) and quantitatively demonstrated improved retention for sialylated species. Examples of these techniques and their applications are reported in this work. | en_US |
| dc.description.note | October 2015 | en_US |
| dc.identifier.citation | Bodnar, E.; Perreault, H. Qualitative and Quantitative Assessment on the use of Magnetic Nanoparticles for Glycopeptide Enrichment. Anal Chem. 2013 Nov 19;85(22):10895-903. doi: 10.1021/ac402332z. Epub 2013 Oct 30 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/30649 | |
| dc.language.iso | eng | en_US |
| dc.publisher | American Chemical Society | en_US |
| dc.rights | open access | en_US |
| dc.subject | Mass Spectrometry | en_US |
| dc.subject | Glycan | en_US |
| dc.subject | Tandem Mass Tag | en_US |
| dc.subject | Workflow | en_US |
| dc.subject | Enrichment | en_US |
| dc.subject | Sugar | en_US |
| dc.subject | Chemistry | en_US |
| dc.subject | Monoclonal Antibody | en_US |
| dc.subject | Immunoglobulin | en_US |
| dc.subject | Sialylation | en_US |
| dc.subject | Pharmaceutical | en_US |
| dc.subject | Herceptin | en_US |
| dc.subject | Trastuzamab | en_US |
| dc.subject | Solid Phase Extraction | en_US |
| dc.subject | Click chemistry | en_US |
| dc.subject | Azido sugar | en_US |
| dc.subject | iTRAQ | en_US |
| dc.subject | MALDI | en_US |
| dc.title | Glycopeptide Enrichment Workflows for Downstream Mass Spectrometric Analysis | en_US |
| dc.type | doctoral thesis | en_US |