High sensitivity (zeptomole) detection of BODIPY heparan sulfate (HS) disaccharides by ion-paired RP-HPLC and LIF detection enables analysis of HS from mosquito midguts.

The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes that control homeostasis and drive development in multicellular animals. In addition, HS is involved in the infection of mammals by viruses, bacteria and parasites. The current detection limit for fluorescently labelled HS disaccharides that is in the low femtomole range (10-15 mol), has effectively hampered investigations of HS composition from small, functionally-relevant populations of cells and tissues. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced fluorescence detection of BODIPY-FI-labelled disaccharides. The method provides an unparalleled increase in the sensitivity of detection by [~] six orders of magnitude, to the zeptomolar range ([~]10-21 moles), enabling detection of <1000 labelled molecules. This facilitates determination of HS disaccharide compositional analysis from minute biological samples, as demonstrated by analysis of HS isolated from the midguts of Anopheles gambiae mosquitoes that was achieved without approaching the limit of detection.