Biology Reference
In-Depth Information
Chapter 43
N
-Glycoprotein Enrichment by Lectin Affi nity
Chromatography
Eliel Ruiz-May , Carmen Catalá , and Jocelyn K.C. Rose
Abstract
Lectins are proteins that bind to sugars with varying specifi cities and several have been identifi ed that show
differential binding to structurally variable glycans attached to glycoproteins. Consequently, lectin affi nity
chromatography represents a valuable tool for glycoproteome studies, allowing enrichment of glycopro-
teins in samples prior to their identifi cation by mass spectrometry (MS). From the perspective of plant
scientists, lectin enrichment has proven useful for studies of the proteomes of the secretory pathways and
cell wall, due to the high proportion of constituent proteins that are glycosylated. This chapter outlines a
strategy to generate samples enriched with glycoproteins from bulk plant tissues prior to further character-
ization by MS, or other techniques.
Key words
Glycoprotein, Lectins, Affi nity chromatography, Concanavalin A
1
Introduction
Glycosylation is a highly complex posttranslational modifi cation
associated with many eukaryotic proteins, involving the attach-
ment of oligosaccharide moieties and their subsequent modifi ca-
tion by a large battery of glycan modifying enzymes. This results in
structurally diverse pool of glycoproteins and glycoforms [
1
,
2
]
.
In plants, these glycoproteins can be classifi ed in
N
-glycoproteins,
where
N
-glycans are covalently linked to asparagine in the sequon
N-X-(S/T), where X can be any amino acid except proline [
3
], and
O
-glycoproteins, where in the glycan is attached to the hydroxyl
group of serine, threonine, or hydroxylated proline residues [
4
-
7
],
with no specifi c sequon. Several analytical platforms have been
developed for systematic studies of glycoproteins from bacteria,
yeast and animals [
8
-
14
], but there are not yet an analogous sys-
tem for plant glycoproteomes. A typical workfl ow might comprise
front-end enrichment of glycoproteins/glycopeptides, identifi ca-
tion of peptide sequence, determination of glycosylation sites and
