Zebrafish (Danio rerio) are an ideal model organism for disorders in various human body systems and neurodegenerative diseases. Despite the wide use of zebrafish as a model and the near complete zebrafish proteome, to date, N-glycosylation in zebrafish has not been studied.1,2 Comparisons of proteins in zebrafish and mammals show that proteins are conserved.3 Further understanding of N-glycosylation in zebrafish could bring important insights for human disease states.
Baycin-Hizal et al.4 sought to characterize N-linked glycoproteins involved in zebrafish development and identify glycosylation sites. Wild-type fertilized zebrafish embryos were prepared by homogenization, and then proteins were extracted, denatured, and digested with two proteases, trypsin and chymotrypsin, to increase the number of glycopeptides.
A technique developed by the Baycin-Hizal group in previous studies, solid-phase extraction of N-linked glycopeptides (SPEG), was used to enrich the N-linked glycopeptides. SPEG technique uses hydrazide chemistry to immobilize sugar containing peptides or proteins on hydrazide beads. The formation of chemical hydrazone bonds between the glycans and beads enables glycosylated peptides to be separated from the non-glycosylated peptides. Then, the captured peptides can be hydrolyzed with glycosidases followed by analysis using mass spectrometry.5,6
Following the use of SPEG, analysis of glycopeptides took place on a LC MS/MS on an LTQ Orbitrap Velos (Thermo Scientific). Samples were analyzed three times to assess the reproducibility and validate the peptides and glycosylation sites discovered.
A total of 855 peptide identifications were assigned using trypsin and chymotrypsin digestion after comparison against the NCBI database and MASCO software. This total included 265 unique glycopeptide sequences and a number of duplicate glycopeptides. Of those 265 sequences, 269 glycosites and 169 N-glycosylated proteins were identified.
The SPEG method was further demonstrated by the discovery of 676 peptides, which included consensus N-X-S/T glycosylation sites. This supported the capability of the SPEG method to enrich the N-linked glycopeptides containing consensus N-linked glycosylation sites.
The identified proteins were categorized into groups based on their functionality. The gene ontology groups included transporters, cell adhesion, ion channels and ion binding, lipid metabolism, development, and receptors. 19.3% of proteins were previously uncharacterized and were placed in the unknown function group.
The Baycin-Hizal group organized their findings into a SQL database, known as the GlycoFish database.7 The GlycoFish database is accessible to anyone interested in zebrafish glycopeptides. This research, as well as the use of the SPEG technique to identify glycopeptides, will enhance the understanding of glycosylation in human disease pathologies. Since the publication of this data, the SPEG technique has already been successfully used in the characterization of glycoproteins in a wide range of model organisms, including Chinese hamster ovary cells, mouse tissues, and Aspergillus niger.8,9,10
References
1. Singh S.K., et al. (2010) ‘Proteomic profile of zebrafish brain based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization MS/MS analysis‘, Zebrafish, 7 (2), (169-177)
2. De Souza, A.G. (2009) ‘Large-scale proteome profile of the zebrafish (Danio rerio) gill for physiological and biomarker discovery studies‘, Zebrafish, 6 (3), (pp. 229-238)
3. Guo, S. (2009) ‘Using zebrafish to assess the impact of drugs on neural development and function‘, Expert Opinion on Drug Discovery, 4 (7), (pp. 715-726)
4. Baycin-Hizal, D., et al. (2011) ‘GlycoFish: A database of zebrafish N-linked glycoproteins identified using SPEG method coupled with LC/MS‘, Analytical Chemistry, 83 (13), (pp. 5296-5303), published online June 8. doi: 10.1021/ac200726q
5. Tian, Y., et al. (2007) ‘Solid-phase extraction of N-linked glycopeptides‘, Nature Protocols, 2 (2), (pp. 334-339)
6. Zhou, Y., et al. (2007) ‘Isolation of N-linked glycopeptides from plasma‘, Analytical Chemistry, 79 (15), (pp. 5826-5837)
7. GlycoFish database. http://betenbaugh.jhu.edu/GlycoFish
8. Baycin-Hizal, D., et al. (2012) ‘Proteomic analysis of Chinese hamster ovary cells‘, Journal of Proteome Research, 11 (11), (pp. 5265-5276)
9. Kaji, H., et al. (2012) ‘Large-scale identification of N-glycosylated proteins of mouse tissues and construction of a glycoprotein database, GlycoProtDB‘, Journal of Proteome Research, 11 (9), (pp. 4553-4566)
10. Wang, L., et al. (2012) ‘Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry‘, Journal of Proteome Research, 11 (1), (pp. 143-156)
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