Publication Information
Song et al., 2013
No external accession available
Abstract
J Proteomics. 2013 Nov 20;93:343-55. doi: 10.1016/j.jprot.2013.07.032. Epub 2013
Aug 27.
N-glycan occupancy of Arabidopsis N-glycoproteins.
Song W(1), Mentink RA, Henquet MG, Cordewener JH, van Dijk AD, Bosch D, America
AH, van der Krol AR.
Author information:
(1)Laboratory of Plant Physiology, Wageningen University and Research Centre,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; Plant Research
International, Wageningen University and Research Centre, Droevendaalsesteeg 1,
6708 PB Wageningen, The Netherlands.
Most secreted proteins in eukaryotes are modified on the amino acid consensus
sequence NxS/T by an N-glycan through the process of N-glycosylation. The
N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to
different mannose-type N-glycans or, when the protein passes through the Golgi
apparatus, to different complex glycan forms. Here we describe the capturing of
N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis
plants and release of these captured peptides following Peptide N-glycosidase
(PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of
peptides released as a consequence of the PNGase treatment was analyzed by two
dimensional nano-LC-MS. As the PNGase treatment of glycopeptides results in the
deamidation of the asparagine (N) in the NxS/T site of the released peptide,
this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation
'signature'. The efficiency of PNGase F and PNGase A in peptide release is
discussed. The identification of proteins with a single glycopeptide was limited
by the used search algorithm but could be improved using a reference database
including deamidated peptide sequences. Additional stringency settings were used
for filtering results to minimize false discovery. This resulted in
identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of
which 28 putative glycoproteins, that were previously not annotated as secreted
protein in The Arabidopsis Information Resource database (TAIR). Furthermore,
the identified glycosylation site occupancy helped to determine the correct
topology for membrane proteins. A quantitative comparison of peptide signal was
made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from
three replicate leaf samples using a label-free MS peak comparison. As an
example, the identified membrane protein SKU5 (AT4G12420) showed differential
glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan
modification on single protein.
BIOLOGICAL SIGNIFICANCE: Proteins that enter the secretory pathway are mostly
modified by N-glycans. The function of N-glycosylation has been well studied in
mammals. However, in plants the function of N-glycosylation is still unclear,
because glycosylation mutants in plants often do not have a clear phenotype.
Here we analyzed which proteins are modified by N-glycans in plants by
developing a glycopeptide enrichment method for plant proteins. Subsequently,
label free comparative proteomics was employed using protein fractions from wild
type and from a mutant which is blocked in modification of the N-glycan into
complex glycans. The results provide new information on N-glycosylation sites on
numerous secreted proteins. Results allow for specific mapping of multiple
glycosylation site occupancy on proteins, which provides information on which
glycosylation sites are protected or non-used from downstream processing and
thus presumably are buried into the protein structure. Glycoproteomics can
therefore contribute to protein structure analysis. Indeed, mapping the
glycosylation sites on membrane proteins gives information on the topology of
protein folds over the membrane. We thus were able to correct the topology
prediction of three membrane proteins. Besides, these studies also identified
limitations in the software that is used to identify single modified peptide per
protein. This article is part of a Special Issue entitled: Translational Plant
Proteomics.
© 2013 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.jprot.2013.07.032
PMID: 23994444 [Indexed for MEDLINE]
