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

Chi-Hung Lin, Chu Wei Kuo, Donald L. Jarvis, Kay Hooi Khoo*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)87-92
Number of pages6
JournalProteomics
Volume14
Issue number1
DOIs
Publication statusPublished - Jan 2014
Externally publishedYes

Keywords

  • C18 RP SPE
  • Glycomics
  • Glycoproteomics
  • MS
  • N-Glycan fractionation

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