Determination of molecular weight profile for a bioactive β-(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.