title: Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.
abstract: Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).
authors.affiliation: †Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Asperger
authors.affiliation: †Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Marx
authors.affiliation: †Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Albers
authors.affiliation: ‡Chelab Silliker, Via Fratta 25, 31023 Resana, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Molin
authors.affiliation: ‡Chelab Silliker, Via Fratta 25, 31023 Resana, Italy.
authors.forename: Odra
authors.initials: O
authors.lastname: Pinato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002853:Chromatography, Liquid; D004529:Egg White / Q000737:chemistry*; D005260:Female; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D009113:Muramidase
keywords: CID; ETD; HEL; LC−MS/MS; MALDI-TOF/TOF; N-glycosylation; bioinformatics; glycan heterogeneity; hen egg white lysozyme; nonconsensus sequon