Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 10 10(3) kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30-2400 kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10 min using only 6 pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.