Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Revealing Cleavage Sites of Chitosan Hydrolases

Abstract

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an O-tag; (iii) quantifying paCOS using [C, H]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

“…The same equipment was used for all LC‐MS n methods described subsequently. Hydrolysis products were analyzed for DP and F A using an UHPLC‐ESI‐MS 1 method which was developed based on the methods described by Cord‐Landwehr et al (). Aliquots of 2 μg of each sample were injected into the LC‐MS n (elution profile in supplement).…”

“…Samples were analyzed using LC‐MS 2 (elution profile in supplement). Results of quantification according to DP and F A and quantitative pattern determination were combined and were evaluated according to Cord‐Landwehr et al ().…”

A Review of the Preparation, Analysis and Biological Functions of Chitooligosaccharide

“…In addition, Cordlandwehr et al developed a promising method using MS which could be used for sequencing and quantitative analysis of partially acetylated COS (pa-COS). Firstly, a deuterated reagent was used to transform pa-COS into fully N -acetylated chitin oligomers; Secondly, complete quantification of pa-COS was analyzed by adding [ 13 C, 2 H]-labelled isotopologs as interior labels in LC-MS; Finally, quantitative measurement of pa-COS by tandem MS could be promoted by using the 18 O-tag to label the reducing end [56]. …”

Chitosan perception inArabidopsisrequires the chitin receptor AtCERK1 suggesting an improved model for receptor structure and function

“…The reactions were stopped by removing the enzymes with a 3K-Pesfilter (VWR, Darmstadt, Germany) at 4ºC, centrifuging at 13000 rpm for 30 min. The target hexamers were analysed using HILIC-ESI-MS 31 .…”

“…Separation of oligomers was achieved using size exclusion chromatography (SEC), as described 30 . Purified oligomers were analyzed using UHPLC-ELSD-ESI-MS 31 .…”