Glycan Quantitation and Glycan Abundance

[Orbitrap_SciLib]

The field of glycomics is transitioning from qualitative to quantitative, where the focus is on the biological insights into the systems being studied. These functional insights often require comparing relative glycan abundance between two or more biological states to elucidate the role of glycans in a disease or to generate lead candidates as part of biomarker discovery programs. Mass spectrometry has emerged as a key technology for the identification of glycans from a wide range of biological sources. Here we describe techniques for relative quantitation of glycans using mass spectrometry.

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Workflow Overview for 2-AA Quantitation

Glycan quantitation has seen a number of method developments in recent years. One such approach takes advantage of reductive amination to introduce stable isotope labels onto the glycans.  Similar to the 2-AB label used in glycan characterization, the 2-aminobenzoic acid (2-AA) label offers many of the same benefits for LC-MS analysis. However, it also available commercially as stable isotopic ¹²[C6]-2-aminobenzoic acid (¹²[C6]-2-AA) and ¹³[C6]-2-AA variants and thus can be used for relative quantitation experiments.^[1]

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References

1. Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan scre...

Prien, J.M., Prater, B.D., Qin, Q. and Cockrill, S.
Anal.  Chem. 2010, 82, 1498-1508.

Sample Preparation

Sample Preparation Workflow for 2-AA Quantitation

Stable isotope labeling with a ¹²[C6] or ¹³[C6] 2-AA fluorescent tag is a method for MS-based quantitation of differential glycan abundances across two different experimental conditions. In stable isotopic 2-AA labeling experiments, glycans are released from proteins by enzymatic means and differentially labeled in parallel with either the ¹²[C6] or ¹³[C6] fluorescent tag. Upon labeling the samples are then combined. All subsequent processing steps can be performed on the combined samples, greatly reducing sample handling variability and resulting in more accurate quantitation. The equimolar differentially labeled paired sample is then subjected to direct infusion or LC-MS-based relative quantitative analysis.

Resources

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screeni...

Prien, J.M., Prater, B.D., Qin, Q. and Cockrill, S.
Anal.  Chem. 2010, 82, 1498-1508.

Mass Spectrometry

Mass Spectrometry Workflow for 2-AA Quantitation

 
Labeling with reductive amination tags such as 2-AA provides a number of benefits for MS analysis: enhanced ionization efficiency in the negative ion mode, labeling under aqueous conditions with no prior sample work-up, and predictable fragmentation of the labeled glycan, which leads to abundant ions that can be assigned unambiguously.

Generally, 2-AA quantitation experiments are performed by LC-MS. Because the glycans are derivatized with isotopic labels, they behave similarly within the separation stage. They co-elute at the same time but can easily be differentiated by a mass spectrometer because of the mass differences of the two isotope labels (6Da between the ¹²[C6] and ¹³[C6] isotopic pair). The relative peak intensities of multiple isotopically distinct glycans are used to determine the average change in glycan abundance in the treated sample. The unmatched resolution and mass accuracy afforded by Orbitrap detection provides discrimination between co-eluting isobaric ions, which is essential for correct 2-AA quantitation.

Since quantitation is dependent upon precursor ions, there is no limit on the types of fragmentation that can be used for MS/MS. Data-dependent CID or HCD can be used for structural elucidation. 

Data Analysis Workflow for 2-AA Quantitation

Data analysis of 2-AA experiments requires both glycan quantitation and structural elucidation.

Quantitation is performed at the MS level by comparing the intensities of the 2-¹²[C6]-AA- and 2-¹³[C6]-AA-labeled precursor ions at high resolution. These ratios can be determined using the PerfectPair workflow exclusive to Thermo Scientific SIEVE software. In this workflow SIEVE™ software enables quantitation of two precursor masses related by a well-defined mass difference within a defined retention time window.
 
For glycan identification and structural elucidation, SimGlycan™ software from Premier BIOSOFT accepts raw data files from Thermo Scientific mass spectrometers and elucidates the associated glycan structure with high accuracy using database searching and scoring techniques.