One of the major goals of glycomics is to characterize the glycosylation differences. Quantification of protein glycosylation is required to distinguish this subtle difference. It is needed for a wide range of reasons, including discovering glycan biomarkers for diagnosis, prognosis, and drug metabolism and pharmacokinetics. The absolute quantification is defined to determine the exact sample concentrations for individual glycans while a relative quantification provides only a percentage of the normalized total glycan content. Absolute quantification is often difficult, because samples are normally analyzed individually by comparison of data from mass spectrometry (MS), or chromatography, where subtle qualitative and/or quantitative differences can easily be missed. As the content of individual glycans is biologically significant for glycan function, absolute glycan quantification is biologically relevant and necessary to provide a full picture of disease related changes of glycosylation. The use of MS in combination with stable isotope labeling for quantitative analysis has become routine in metabolomics and proteomics. It is has been employed for the quantitative analysis in glycomics.
Stable isotope mediated glycan quantification can be performed by spiking isotope enriched standards into the sample or by labeling the entire glycan mixture with a heavy-isotope labeled tag.
1. Stable isotope labeled tags
Due to a lack of heavy-isotope labeled glycan standards, heavy-isotope based strategies for glycan quantification were mainly focused on the incorporation of stable isotope labeled tags into the glycan samples for differential quantitative glycan analysis. An effective method is to permethylate glycans using different isotopically labeled methyl iodide reagents. This method has been expanded using singly, doubly, and triply deuteriated methyl iodide agent, thus allowing tetraplexing. This procedure has been used to analyze N-linked glycans released from various mixtures of glycoproteins.
Figure 1. Representation of multiplexed comparative glycomic mapping through permethylation using stable isotopic methyl iodide reagents
2. Absolute glycan quantification using stable isotope glycan as internal standards
The significant mass increment of the isotopically labeled standard over the natural compound gives rise to a clear separation of the isotopic profiles of analyte and standard which is important for glycan quantification. The use of stable-isotope labeled glycans as internal standards allow the absolute quantification of even low abundant glycan markers in complex mixtures. It is important as well for the development of absolute quantification of glycans as clinical biomarkers. Absolute glycan quantification using stable isotope glycan as internal standards also should help to rapidly establish methods employing isotopic dilution for glycan disease marker quantification.
Figure 2. Quantitative MALDI-TOF MS spectra of IgG1 glycan profile in the presence of stable isotope labelled internal N-glycan standards (marked with an asterisk)1
There are other methods for the absolute glycan quantification, such as RapiFluor-MS labeled glycan standards for the glycan quantification, and 2-Aminobenzamide labeled glycans using HPLC with fluorescence detection.
1. B. Echeverria, J. Etxebarria, N. Ruiz, Á. Hernandez, J. Calvo, M. Haberger, D. Reusch, N.-C. Reichardt (2015). Chemo-enzymatic synthesis of 13C labeled complex N-glycans as internal standards for the absolute glycan quantification by Mass Spectrometry, Anal. Chem., 87, 11460-11467.
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