Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry

Abstract

Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps.

Cyclodextrin-Based Functional Glyconanomaterials

“…In the last decade, however, multivalent polycations based on molecular systems emerged as a promising alternative [86][87][88][89][90][91][92][93][94][95][96]. Polycationic cyclodextrins have become the iconic representatives in this category [5,[97][98][99][100][101][102][103][104]. The efficiency in compacting nucleic acids into nanocomplexes (so-called CDplexes) capable of mediating cell entry and subsequent expression of the nucleic acid anticipated activity (transfection) was significantly enhanced for prototypes that can additionally establish cyclodextrin-cyclodextrin interactions through either hydrophobic , host-guest [79,134] or aromatic-aromatic contacts [135][136][137][138][139][140][141][142][143][144][145][146].…”

Direct Analysis in Real Time Mass Spectrometry for Characterization of Large Saccharides

“…The enzymes required to hydrolyze the polysaccharides are expensive and mostly unavailable, and the digestion methods normally takes hours if not longer. 17,18 Analytical techniques including thin layer chromatography, liquid chromatography (LC), 19,20 gas chromatography (GC), 21,22 or LC coupled to mass spectrometry (LC-MS) 23,24 are then used for the identification of saccharide hydrolysates. Until now, the majority of studies have focused on the determination of monomer composition and acquisition of fingerprints of polysaccharides on the basis of small digested products.…”

“…Mass spectrometry is a powerful tool that can be used for compound identification in many applications. 22,24,30 However, direct infusion of large polysaccharides using traditional ion source such as electrospray ionization (ESI) is difficult due to inefficient ionization and mass range limitation. LC in combination with MS can provide much better sensitivity for the analysis of the digested mixtures because MS measures m/z values for every single compound eluted from LC.…”

“…Usually, large polysaccharide is first digested into small molecules through chemical, physical, or enzymatic methods, − and the products are then analyzed by other analytical techniques. The enzymes required to hydrolyze the polysaccharides are expensive and mostly unavailable, and the digestion methods normally takes hours if not longer. , Analytical techniques including thin layer chromatography, liquid chromatography (LC), , gas chromatography (GC), , or LC coupled to mass spectrometry (LC-MS) , are then used for the identification of saccharide hydrolysates. Until now, the majority of studies have focused on the determination of monomer composition and acquisition of fingerprints of polysaccharides on the basis of small digested products. − Monomer composition is determined by matching the known monosaccharide standards such as glucose and mannose with the complete hydrolysis products with LC .…”

“…To set up the evaluation standards and compare the differences among various plant polysaccharides, liquid chromatographic profiles of digested fragments of Chinese herb polysaccharides were considered as the fingerprints for quality control. , Sugar hydrolysates with similar or isomeric carbohydrates can coelute in LC due to its limited resolution, and impurities or unknowns cannot be identified using LC alone. Mass spectrometry is a powerful tool that can be used for compound identification in many applications. ,, However, direct infusion of large polysaccharides using traditional ion source such as electrospray ionization (ESI) is difficult due to inefficient ionization and mass range limitation. LC in combination with MS can provide much better sensitivity for the analysis of the digested mixtures because MS measures m / z values for every single compound eluted from LC. , However, these methods involve tedious multistep sample preparation (saccharide extraction, purification, and hydrolysis), and method development and optimization are typically time-consuming and labor-intensive because polar carbohydrates show close to no retention on reverse phase columns.…”