Determination of molecular weight profile for a bioactive beta-(1-->3) polysaccharides (Curdlan)

This paper focuses on the development of methodology based on MALDI-TOF mass spectrometry for evaluation of molecular weight profile of the water-insoluble portion of an extracellular polysaccharide, i.e. Curdlan. As previously demonstrated, MALDI analysis of water-insoluble Curdlan fraction gave number-average (M(n)) and weight-average (M(w)) molecular weights of 8000 and 8700 Da, respectively [T.W.D. Chan, K.Y. Tang, Rapid Commun. Mass Spectrom. 17 (2003) 887]. To validate the MALDI determined molecular weight information, several additional analytical schemes were used to analysis the water-insoluble Curdlan fraction. In all cases, the water-insoluble Curdlan sample was fractionated by gel permeation chromatography (GPC) using Sephadex G-75 column. The M (n) of low-mass and narrow distributed polysaccharide fractions were obtained by MALDI-MS. Good linearity was found in the calibration plot constructed from the measured M (n)-values and the corresponding elution time/volume. The relative quantity of various fractionated samples was then measured using three different approaches. These include (a) direct refractometric analysis; (b) UV-vis absorption analysis of the Aniline Blue stained sample; and (c) GC-MS analysis of the hydrolyzed and TMS-derivatized sample. Using results obtained from theses quantification methods and the correlation function between the GPC retention time and M (n), the MW and MWD of water-insoluble Curdlan were obtained. Our results demonstrated that the previous use of MALDI methods for measuring M(n), M(w) and polydispersity (PD) of water-insoluble Curdlan (with and without GPC fractionation) were unreliable. However, by standardizing the narrow distributed polysaccharides using MALDI-MS method, reliable molecular weight information for dispersed polysaccharides could be obtained. The M (n),M (w) and PD of the water-insoluble Curdlan were found to be 22,000, 31,500 Da and 1.40, respectively.