A Multidimensional Mass Spectrometry-Based Workflow for De Novo Structural Elucidation of Oligosaccharides from Polysaccharides.

Carbohydrates play essential roles in a variety of biological processes that are dictated by their structures. However, characterization of carbohydrate structures remains extremely difficult and generally unsolved. In this work, a de novo mass spectrometry-based workflow was developed to isolate and structurally elucidate oligosaccharides to provide sequence, monosaccharide compositions, and glycosidic linkage positions. The approach employs liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods in a 3-dimensional concept: one high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) analysis for oligosaccharide sequencing and two ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ MS) analyses on fractionated oligosaccharides to determine their monosaccharides and linkages compositions. The workflow was validated by applying the procedure to maltooligosaccharide standards. The approach was then used to determine the structures of oligosaccharides derived from polysaccharide standards and whole food products. The integrated LC-MS workflow will reveal the in-depth structures of oligosaccharides.

A nonenzymatic method for cleaving polysaccharides to yield oligosaccharides for structural analysis.

Polysaccharides are the most abundant biomolecules in nature, but are the least understood in terms of their chemical structures and biological functions. Polysaccharides cannot be simply sequenced because they are often highly branched and lack a uniform structure. Furthermore, large polymeric structures cannot be directly analyzed by mass spectrometry techniques, a problem that has been solved for polynucleotides and proteins. While restriction enzymes have advanced genomic analysis, and trypsin has advanced proteomic analysis, there has been no equivalent enzyme for universal polysaccharide digestion. We describe the development and application of a chemical method for producing oligosaccharides from polysaccharides. The released oligosaccharides are characterized by advanced liquid chromatography-mass spectrometry (LC-MS) methods with high sensitivity, accuracy and throughput. The technique is first used to identify polysaccharides by oligosaccharide fingerprinting. Next, the polysaccharide compositions of food and feces are determined, further illustrating the utility of technique in food and clinical studies.

Development of an Extensive Linkage Library for Characterization of Carbohydrates.

The extensive characterization of glycosidic linkages in carbohydrates remains a challenge because of the lack of known standards and limitations in current analytical techniques. This study encompasses the construction of an extensive glycosidic linkage library built from synthesized standards. It includes an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of glycosidic linkages derived from disaccharides, oligosaccharides, and polysaccharides present in complicated matrices. We present a method capable of the simultaneous identification of over 90 unique glycosidic linkages using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ MS) operated in dynamic multiple reaction monitoring (dMRM) mode. To build the library, known monosaccharides commonly found in plants were subjected to partial methylation to yield partially derivatized species representing trisecting, bisecting, linear, and terminal structures. The library includes glycosidic linkage information for three hexoses (glucose, galactose, and mannose), three pentoses (xylose, arabinose, and ribose), two deoxyhexoses (fucose and rhamnose), and two hexuronic acids (glucuronic acid and galacturonic acid). The resulting partially methylated monosaccharides were then labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by separation and analysis by UHPLC/dMRM MS. Validation of the synthesized standards was performed using disaccharide, oligosaccharide, and polysaccharide standards. Accuracy, reproducibility, and robustness of the method was demonstrated by analysis of xyloglucan (tamarind) and whole carrot root. The synthesized standards represent the most comprehensive group of carbohydrate linkages to date.

Strategy for Structural Elucidation of Polysaccharides: Elucidation of a Maize Mucilage that Harbors Diazotrophic Bacteria.

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

Liquid Chromatography-Tandem Mass Spectrometry Approach for Determining Glycosidic Linkages.

The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 µg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reversed-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15 min run time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan ( Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.

Revisiting monosaccharide analysis - quantitation of a comprehensive set of monosaccharides using dynamic multiple reaction monitoring.

A rapid method for the quantitation of sixteen neutral and acidic monosaccharides, from both animal and plant sources was developed using ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC/QqQ-MS) in dynamic multiple reaction monitoring (dMRM) mode. Monosaccharides including three pentoses (ribose, xylose, arabinose), two deoxyhexoses (rhamnose, fucose), five hexoses (fructose, mannose, allose, glucose, galactose), two hexuronic acids (glucuronic acid, galacturonic acid), and two N-acetyl-hexosamines (GlcNAc, GalNAc), were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), while underivatized sialic acids, Neu5Ac and Neu5Gc, were simultaneously analyzed with a 10-minute run. With the optimized UHPLC conditions, baseline separations of the isomers were achieved. The sensitivity and calibration ranges of this method were determined. The limits of detection were between femtomoles and attomoles with linear ranges spanning four to six orders of magnitude and coefficients of variation (CVs) ≤7.2%. Spiking experiments performed on a pooled fecal sample demonstrated the high accuracy of this method even when applied to samples with complicated matrices. The validated method was applied to fecal samples from an infant transitioning from breast milk to weaning foods. Major milk monosaccharides including galactose, fucose, glucose, GlcNAc, and Neu5Ac were found to be the most abundant components in the feces of milk-fed infants. PMP-derivatives of nine other monosaccharides including apiose, lyxose, altrose, talose, gulose, glucosamine, galactosamine, mannosamine, and N-acetylmannosamine (ManNAc) were also tested and could be added to the quantitation method depending on the need. The speed and sensitivity of the method makes it readily adaptable to rapid throughput analysis of monosaccharides in biological samples.

Changes in cellular glycosylation of leukemia cells upon treatment with acridone derivatives yield insight into drug action.

A new acridone derivative 2-aminoacetamido-10-(3, 5-dimethoxy)-benzyl-9(10H)-acridone hydrochloride (8a) has been shown to have potent antitumor activity. In order to understand the underlying action mechanism of 8a, three compounds of the same class with structures optimized step-by-step, 9(10H)-acridone (A), 10-(3,5-dimethoxy) benzyl-9(10H)-acridone (I) and 8a, were exposed to CCRF-CEM leukemia cell to determine the N-glycosylation changes using the microfluidic HPLC-chip-TOF MS platform. N-Glycans from whole cell lysates (WCL) and cell membranes (CM) were analyzed using isomer-sensitive chip-based porous graphitized carbon nano-LC/MS. A total of 223 N-glycan compositions and 398 N-glycan compounds were identified. Comparison of the two analyses showed that more apparent changes were observed in the CM compared with WCL, suggesting that CM may be a more sensitive indicator of changes in glycosylation. Upon 8a exposure to CCRF-CEM cells, a significant decrease in high-mannose-type glycans was observed. Different expressions of oligosaccharyltransferase subunits appear to play a key functional role in regulating the hypoglycosylation and contribute to the action mechanism of 8a. Taken together our findings suggest that glycosylation is strongly affected by therapeutic potency and can be used as possible biomarkers for monitoring toxicity and antitumor activity of 8a.