Biology Reference
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such as sialic acid residues, which are detected in mammalian
glycoproteins, have not been registered in the KEGG Glycan [ 6 ],
GlycoMod Tool [ 7 ], and GlycoSuite [ 8 ] databases.
In higher plant cells, the transfer of Glc 3 Man 9 GlcNAc 2
precursor glycan to the nascent polypeptide, and the modifi cation
of the glycan chain, occurs through a conserved biosynthetic
pathway [ 9 ]. A set of glucosidase and mannosidase converts
Glc 3 Man 9 GlcNAc 2 to Man 8 GlcNAc 2 sequentially in the endoplas-
mic reticulum, and the Man 8 GlcNAc 2 glycan is then trimmed to
Man 5 GlcNAc 2 by cis -Golgi-resident mannosidase I [ 10 ], and fur-
ther modifi ed into complex chains by Golgi mannosidase II and
glycosyltransferases. It has been reported that N -acetylglucosaminyl-
transferase I and N -acetylglucosaminyltransferase II localize in the
cis -Golgi to medial-Golgi compartments; and
β
1,2-xylosyl-
transferase,
α
1,3-fucosyltransferase,
α
1,4-f
α
α1,4-fαcosyltransferase and
β
1,3-galactosyltransferase localize in the medial-Golgi to trans -
Golgi compartments [ 9 ]. Interestingly, recent investigations have
provided evidence on the traffi cking of glycoproteins [ 11 - 15 ] to
the plastids through the secretory pathway. There is probably con-
siderable membrane traffi c between the endomembrane system
and the plastids.
However, the physiological importance of glycan modifi cation
of glycoproteins in plants remains obscure. To clarify this, it is nec-
essary to employ a rapid, effi cient, sensitive and high-throughput
method for analyzing plant N-linked oligosaccharide chains. Here,
we describe an application of the glycoblotting-mass spectrometry
method [ 16 - 20 ] that has made comprehensive N -glycome analysis
possible in recent years.
2
Materials
Milli-Q water is used in all solutions containing water. The metha-
nol and acetonitrile (ACN) are LC-MS grade.
1. 50 mM citrate-phosphate buffer, pH 5: Dissolve 0.48 g of citric
acid and 0.39 g of sodium dihydrogen phosphate dehydrate in
about 50 mL of water, and adjust pH with NaOH (1 N). Make
up to 50 mL with water. Store at room temperature.
2.1 Sample
Preparation
2. 100 mM (±)-dithiothreitol (DTT): Dissolve 1.54 g of DTT in
10 mL of water to prepare 1 M solution. Store at −20 °C.
Dilute to 100 mM before use.
3. 100 mM 2-iodoacetamide: Dissolve 0.18 g of 2-iodoacetamide
in 1 mL of water. Prepare before use.
2.2 Hydrolysis
of N -Glycans
1. Protease K (0.6 U/
L, Roche, Basel, Switzerland): Store at 4 °C.
2. Glycopeptidase A (50
μ
μ
U/
μ
L, Roche): Store at 4 °C.
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