Glycosylation profiling of dog serum reveals differences compared to human serum

Abstract

Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG – the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.

Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis

“…We have previously reported the detailed glycosylation analysis of canine serum 38 . To investigate the impact of filarial infection by D. immitis on the total N-glycosylation profile of serum, we analyzed serum samples from two different dog cohorts.…”

“…N-glycans were released from serum or IgG using Rapid PNGase F and fluorescently labeled with procainamide as described previously 38 . The procainamide labeled N-glycans were analyzed by HILIC-UPLC with fluorescence and mass detection on a Waters Acquity H-class instrument composed of a binary solvent manager, a sample manager, a fluorescence detector (excitation wavelength 310 nm; detection wavelength 370 nm) and a QDa mass detector (settings: positive mode; target sampling rate: 10 point/sec; gain: 1; capillary voltage: 1.5 kV; probe temperature: 600 °C).…”

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

“…After reconstitution in 100 µL of water, two 20 µL aliquots were deglycosylated with Rapid PNGase F (NEB #P0710) following manufacturer's instructions (2-step protocol that includes a pre-incubation at 80 °C for 2 min in Rapid PNGase buffer for complete deglycosylation). Released N-glycans were labeled with procainamide [4-amino-N-(2-diethylaminoethyl) benzamide] according to a previously described procedure [24].…”

“…Samples were analyzed using a BEH-XBridge column, (130 Å, 2.5 µm, 2.1 mm x 150 mm; Waters) on a Dionex UltiMate® HPLC with fluorescent detection, in line with a LTQ™ Orbitrap Velos™ Spectrometer (HESI-II probe), using optimized settings for positive mode detection as described before [24]. Structures were assigned based on retention time, m/z (parent ion), and in accordance with known biosynthetic pathways.…”

“…Isolated p36 and p34 were denatured and treated with peptide-N-glycosidase F (PNGase F) that releases the N-linked chains and converts asparagine into aspartic acid. The released N-glycans were labeled with procainamide according to a recently described highly sensitive derivatization method for glycan labeling and analyzed as described in Materials and methods [24]. In Fig.…”