GlycoFish: A Database of Zebrafish N-linked Glycoproteins Identified Using SPEG Method Coupled with LC/MS

Abstract

Zebrafish (Danio rerio) is a model organism to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases, chymotrypsin and trypsin, into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.

Identification and Validation of Atypical N-Glycosylation Sites

“…Compared to previously reported atypical N -glycosites that were identified on the basis of the deamidation of asparagine residues after PNGase F treatment, 4,5 this study further validated the identified atypical motif glycosites by directly identifying their intact glycopeptides. Since the deamidation (N) can occur naturally and during sample preparation, direct identification of the intact glycopeptides with glycan attached is particularly important for confirmation of these atypical N -glycosylation sites detected by mass spectrometry-based glycoproteomics.…”

“…Except for the N-X-C motif, which has been confirmed in several known glycoproteins, 3 all other atypical motifs were only identified on the basis of the deamidation of asparagine (N) residues in the peptides after PNGase F treatment (with/without 18O labeling) using mass spectrometry-based glycoproteomics. 4,5 However, the atypical sites identified on the basis of deamidation (N) are potentially false positives as deamidation (N) could occur naturally or be induced during sample preparation. 6,7 …”

Proteomic Analysis of Chinese Hamster Ovary Cells

“…The lysates (800 μg) were denatured, reduced, alkylated and digested as previously explained 22 . Following tryptic digestion, the samples were desalted through C18 columns and were oxidized with 10 mM sodium periodate at room temperature in dark for 1 hour.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…A database for Drosophila N-linked glycopeptides (http://betenbaugh.jhu.edu/GlycoFly) named GlycoFly has been established as a resource for study of neurological disorders (Baycin-Hizal et al, 2011a). GlycoFish is a database of zebrafish N-linked glycoproteins (Baycin-Hizal et al, 2011b) and can be found at (http://betenbaugh.jhu.edu/ GlycoFish).…”