Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I.
Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quantification by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with (13)C-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with (12)C or (13)C methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as alpha-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of flight MS and electrospray ionization with ion cyclotron resonance-Fourier transformation MS. The measured (12)C:(13)C ratios from mixtures of glycans permethylated with either (12)CH(3)I or (13)CH(3)I were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.
Alvarez-Manilla
Gerardo
G
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA. alvarez@uga.edu
Warren
Nicole Lynn
NL
Abney
Trina
T
Atwood
James
J
Azadi
Parastoo
P
York
Will S
WS
Pierce
Michael
M
Orlando
Ron
R
D000465:Algorithms; D002236:Carbohydrate Conformation; D002247:Carbon Isotopes / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D006847:Hydrocarbons, Iodinated / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008895:Milk, Human; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D011134:Polysaccharides; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002247:Carbon Isotopes; D006023:Glycoproteins; D006847:Hydrocarbons, Iodinated; D009844:Oligosaccharides; D011134:Polysaccharides; C014055:methyl iodide