title: Facile removal of high mannose structures prior to extracting complex type N-glycans from de-N-glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping.
abstract: The relative amount of high mannose structures within an N-glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco-epitopes. An efficient method to separate these two classes of N-glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow-through fraction when peptide-N-glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N-glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de-N-glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N-glycans on C18 resin is dependent not only on size but also increased by the presence of α6-fucosylation. This was shown by comparing the resulting N-glycomic profiles of the washed and low-ACN eluted fractions derived from both a human cancer cell line and an insect cell line.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Department of Molecular Biology, University of Wyoming, Laramie, WY, USA.
authors.forename: Chi-Hung
authors.initials: CH
authors.lastname: Lin
authors.forename: Chu-Wei
authors.initials: CW
authors.lastname: Kuo
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
mesh_terms: D002240:Carbohydrate Sequence; D054794:Glycomics / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry
chemical_list: D006020:Glycopeptides; D008358:Mannose
keywords: C18 RP SPE; Glycomics; Glycoproteomics; MS; N-Glycan fractionation