Determination and Characterization of Site-Specific N-Glycosylation Using MALDI-Qq-TOF Tandem Mass Spectrometry:  Case Study with a Plant Protease

MALDI tandem mass spectrometry analysis on a hybrid quadrupole−quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a nov...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2006-02, Vol.78 (4), p.1093-1103
Main Authors: Bykova, Natalia V., Rampitsch, Christof, Krokhin, Oleg, Standing, Kenneth G., Ens, Werner
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analytical chemistry
Case studies
Chemistry
Chromatographic methods and physical methods associated with chromatography
Chromatography, High Pressure Liquid
Electrophoresis, Polyacrylamide Gel
Exact sciences and technology
General, instrumentation
Glycopeptides - chemistry
Glycoproteins
Glycosylation
Isoelectric Focusing
Lycopersicon esculentum - enzymology
Mass spectrometry
Molecular Sequence Data
Other chromatographic methods
Peptide Hydrolases - chemistry
Peptides
Proteases
Spectrometric and optical methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Tomatoes
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Summary:MALDI tandem mass spectrometry analysis on a hybrid quadrupole−quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac0512711