title: Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry.
abstract: Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Uma K
authors.initials: UK
authors.lastname: Aryal
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Dai
authors.forename: Alisa C
authors.initials: AC
authors.lastname: Mason
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.forename: Zhi-Xin
authors.initials: ZX
authors.lastname: Tian
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Zhou
authors.forename: Dian
authors.initials: D
authors.lastname: Su
authors.forename: Karl K
authors.initials: KK
authors.lastname: Weitz
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Scott E
authors.initials: SE
authors.lastname: Baker
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006834:Hydrazines / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins; D005656:Fungal Proteins; D006023:Glycoproteins; D006834:Hydrazines