Separations of Carbohydrates with Noncovalent Shift Reagents by Frequency-Modulated Ion Mobility-Orbitrap Mass Spectrometry.

An increased focus on characterizing the structural heterogeneity of carbohydrates has been driven by their many significant roles in extant life and potential roles in chemical evolution and the origin of life. In this work, multiplexed drift tube ion mobility-Orbitrap mass spectrometry methods were developed to analyze mixtures of disaccharides modified with noncovalent shift reagents. Since traditional coupling of atmospheric pressure drift tube ion mobility cells with Orbitrap mass analyzers suffers from low duty cycles (<0.1%), a frequency modulation scheme was applied to improve the signal-to-noise ratios (SNR). Several parameters such as the resolution setting and maximum injection time of the Orbitrap analyzer and the magnitude and duration of the frequency sweep were investigated for their impact on the sensitivity gains and resolution of disaccharide-shift reagent adducts. The sweep time and disaccharide concentration had a positive correlation with SNR. The magnitude of the frequency sweep had a negative correlation with SNR. However, increasing the frequency sweep improved the resolution of mixtures of disaccharide analytes. Application of frequency-modulated ion mobility-Orbitrap mass spectrometry to four noncovalently modified glucose dimers allowed for the differentiation of three out of these four analytes.

Organic acid shift reagents for the discrimination of carbohydrate isobars by ion mobility-mass spectrometry.

Carbohydrates are the most abundant class of biomolecules on Earth with a diverse array of biological functions. It is hypothesized that they likely had an important role in the development of life on the primoridal Earth as well. Since sugars have a variety of possible isobaric structures, it is necessary to characterize oligosaccharides beyond their molecular weight. Ion mobility-mass spectrometry (IM-MS) is a promising characterization technique for this purpose, as it is based on differences in charge and collision cross section (CCS). This study reports on the use of new noncovalent ligands as shift reagents to aid in the IM separations of disaccharides. A variety of organic acids were tested as shift reagents with traveling wave IM with the most promising ones being further investigated by drift tube IM. Drift tube IM provided higher resolution separations for the large majority of disaccharide complexes studied. Combining CCS results of the two most promising shift reagents allowed for the complete differentiation of all eight disaccharide standards examined in this study.

The Oligomerization of Glucose Under Plausible Prebiotic Conditions.

The prebiotic origin of polysaccharides, the largest class of biopolymers by mass in extant biology, has seldom been investigated experimentally. Herein, we report on the acid-catalyzed condensation of aqueous solutions of glucose, a model monosaccharide, under plausible prebiotic conditions employing a wet-dry (night-day) protocol with 0.01 M HCl at 50 °C. This protocol leads to the formation of oligosaccharides containing up to eight monomeric units identified by high resolution mass spectrometry. The regio- and stereochemistry of the oligomeric acetal linkages, as well as the quantitative analysis of glucose conversion, are elucidated by combining 1H, 13C and 2D NMR spectroscopy. Ten out of eleven possible acetal linkages, including α- and ß- anomers, have been identified with the α- and ß- 1,6-acetals being the dominant linkages observed. In addition, the acid-catalyzed oligomerization of several glucose disaccharides such as cellobiose, maltose, and gentiobiose are presented along with an accompanying comparison with the corresponding oligomerization of glucose.

Carbohydrate isomer resolution via multi-site derivatization cyclic ion mobility-mass spectrometry.

Oligosaccharides serve many roles in extant life and may have had a significant role in prebiotic chemistry on the early Earth. In both these contexts, the structural and isomeric diversity among carbohydrates presents analytical challenges necessitating improved separations. Here, we showcase a chemical derivatization approach, where 3-carboxy-5-nitrophenylboronic acid (3C5NBA) is used to label vicinal hydroxyl groups, amplifying the structural difference between isomers. We explore the applicability of state-of-the-art ion mobility - mass spectrometry (IM-MS) instrumentation in the analysis of derivatized carbohydrates. In particular we focus on the resolving power required for IM separation of derivatized isomers. A recently developed cyclic ion mobility (cIM) mass spectrometer (MS) was chosen for this study as it allows for multi-pass IM separations, with variable resolving power (Rp). Three passes around the cIM (Rp ∼ 120) enabled separation of all possible pairs of four monosaccharide standards, and all but two pairs of eight disaccharide standards. Combining cIM methodology with tandem mass spectrometry (MS/MS) experiments allowed for the major products of each of the 3C5NBA carbohydrate derivatization reactions to be resolved and unequivocally identified.

Rapid resolution of carbohydrate isomers via multi-site derivatization ion mobility-mass spectrometry.

Identifying small sugar isomers can be challenging by ion mobility-mass spectrometry (IM-MS) alone due to their small collision cross section differences. Herein, we report IM-MS results for multi-site, covalent carbohydrate derivatization with 3-carboxy-5-nitrophenylboronic acid (3C5NBA). Following reaction in aqueous solutions at room temperature, 3C5NBA reacts with each mono- or disaccharide molecule to yield products that each have a distinguishable mobility signature. The reaction was rapid and resulted in the detection of products within 5 min after 3C5NBA was mixed with the analyte. Eight disaccharides that varied in linkage, composition, and configuration (α or ß) as well as four monosaccharides, d-glucose, d-galactose, d-mannose and d-fructose, were included in this study. The derivatives' drift times showed significant shifts, with up to 3-fold gains in resolution when compared to previous literature reports. Moreover, the specific MS/MS fragmentation information gathered from these sugar derivatives provided further validation of the isomers' structures.