Hi all, apologies if these are very basic questions, but I'm fairly new to FACE.
First, I've seen multiple papers using FACE to separate disaccharides. It is possible to separate chondroitin-4-sulphate and chondrotin-6-sulphate disaccharides, however, I do not understand how this is possible. Both GAG chains are are mono-sulphated, so mass to charge ratio is the same, anionic potential is the same, etc. So how are they separated?
Second, and again sorry if this is basic question, but why do disaccharides migrate in "reverse" order, such that higher molecular weight disaccharides are lower on the gel and lower m.w disaccharides are higher? Eg, ΔHA migrates slower and is at the top of the gel at m.w of 379.3, whereas ΔCS-4S has a m.w of 459.4 and is lower on the gel (m.w data from Osago et al., 2014).
Thank you all very much.
Fluorophore-assisted carbohydrate electrophoresis (FACE) separates glycosaminoglycans (GAGs) based on differences in their fluorescent properties, which are influenced by the number and position of sulfate groups. While chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) both have one sulfate group per disaccharide unit, the position of this sulfate group differs between the two molecules. Specifically, C4S has a sulfate group at position 4 of the N-acetylgalactosamine (GalNAc) unit, while C6S has a sulfate group at position 6. This difference in sulfation pattern affects the fluorescence properties of the two molecules, allowing them to be separated by FACE.
Regarding the migration order of disaccharides on the gel, it is influenced by the size and charge of the molecule, as well as the charge and composition of the gel matrix. In FACE, the gel matrix is composed of polyacrylamide crosslinked with bis-acrylamide, and the electrophoresis is run under denaturing conditions (e.g. high temperature, low pH), which can influence the migration order of the disaccharides. In general, smaller and more highly charged molecules will migrate faster and farther through the gel, while larger and less charged molecules will migrate slower and remain closer to the loading well. Therefore, the "reverse" migration order observed in FACE likely reflects the combined influence of molecule size, charge, and gel matrix composition on the electrophoretic mobility of the disaccharides.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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