A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry

Saskia R. Karg, Alexander D. Frey*, Claudia Ferrara, Daniel K. Streich, Pablo Umaña, Pauli T. Kallio

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

14 Citations (Scopus)

Abstract

The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5 kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24 h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5 μg of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method.

Original languageEnglish
Pages (from-to)160-166
Number of pages7
JournalPlant Physiology and Biochemistry
Volume47
Issue number2
DOIs
Publication statusPublished - Feb 2009
MoE publication typeA1 Journal article-refereed

Keywords

  • Horseradish peroxidase
  • MALDI-TOF MS
  • Nicotiana tabacum
  • Peptide-N-glycosidase A
  • Plant N-linked glycosylation

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