Determination of molecular weight profile for a bioactive β-(1 → 3) polysaccharides (Curdlan) Original Research Article

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 image and weight-average image 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 image 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 image-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 image, the MW and MWD of water-insoluble Curdlan were obtained. Our results demonstrated that the previous use of MALDI methods for measuring image 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 image and PD of the water-insoluble Curdlan were found to be 22,000, 31,500 Da and 1.40, respectively.