Published: May 22nd, 2016
This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.
Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.
Glycolipids, glycoproteins, proteoglycans, and glycosaminoglycans constitute the four main classes of complex, heterogeneous carbohydrates collectively known as glycans. As ubiquitous and integral components of the plasma membrane, glycocalyx, and extracellular matrix and fluids, glycans partake in such diverse biochemical processes as endocytosis, intracellular trafficking, cell motility, signal transduction, molecular recognition, receptor activation, cell adhesion, host-pathogen interaction, intercellular communication, immunosurveillance, and immune response initiation.1 Present in nearly every domain of life, enzymes known as glycosyltransferases that ....
Caution: Avoid skin/eye contact with any of the reagents used in this experiment. Upon exposure, thoroughly flush the affected area with water and seek immediate medical advice.
1. Permethylation and Glycan Extraction
A total ion current chromatogram (TIC) showing successful permethylation, hydrolysis, reduction, and acetylation of human blood plasma samples relative to cases in which two critical permethylation steps were executed incorrectly are shown in Figure 3.
Absolute Yield of HexNAcs Relative to Hexoses:
N-acetylhexosamine (HexNAc) partiall.......
In general, the successful production of partially methylated alditol acetates (PMAAs) from hexoses is fraught with fewer difficulties and is more robust than the successful production of N-acetylhexosamine (HexNAc) PMAAs. The exact mechanism behind this phenomenon as it plays out in every step of this procedure is unknown, but must relate to the unique chemistry of the N-acetyl group (rather than hydroxyl group) that is unique to HexNAcs relative to hexoses. The mechanism behind this phenomenon as it r.......
This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.....
|Sodium hydroxide beads
|20-40 mesh, reagent grade 97%
|0.9 mL Spin column
|Pierce division of ThermoFisher Scientific
|Includes plugs and polyethylene frits
|GC-MS autosampler vial (silanized)*
|Target DP High Recovery Vial, 1.5 mL, 12 x 32 mm, Includes Teflon-lined pierceable caps
|1.5 mL polypropylene test tubes
|2 mL polypropylene test tubes
|Dimethyl Sulfoxide (DMSO)
|BioReagent for molecular biology, reagent grade >99.0%
|Contains copper as stabilizer, ReagentPlus 99%
|75002436: Sorvall Legend Micro 17 Centrifuge
|24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424
|13 x 100 glass test tube (silanized)*
|13 x 100 mm borosilicate glass test tubes with screw-cap finish
|Caps for glass test tubes
|Kimble™ Black Phenolic Screw Caps; 13mm-415 GPI thread; PTFE-faced rubber liner.
|Macron Fine Chemicals
|99% purified by redistillation for protein sequencing
|Ammonium hydroxide solution
|NH3 content: 28.0 - 30.0%
|Honeywell Burdick & Jackson
|Plastic vacuum desiccator
|Any model of adequate size
|Stabilized HPLC grade
|Heated evaporation manifold (main unit)
|Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function
|Heated evaporation manifold (overhead evaporator)
|ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module
|Aluminum sample-holder blocks for evaporation manifold
|Block, Aluminum, Reacti-Block S-1, Holds 13mm dia test tubes, 13 holes (14 dia. x 45mm deep)
|Equipped with CTC PAL autosampler
|GCT Premier (Time-of-Flight)
|Split-mode liner (deactivated / silanized)
|Containing a small plug of silanized glass wool
|DB-5ms GC column
|30 m x 0.25 mm ID x 0.25 micron film thickness
|Glass vacuum desiccator (for glassware silanization)
|12" wide; 230 mm plate size
|*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator.
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