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.

A targeted liquid chromatography mass spectrometry method for routine monitoring of cell culture media components for bioprocess development.

The analysis of cell culture media (CCM) components is critical for understanding cell growth kinetics and overall product quality during biomanufacturing. Given the diverse physical and chemical nature of CCM compounds present at a wide range of concentrations, there is an increasing demand for single-platform analytical assays with exceptional specificity and sensitivity. This study presents a targeted LC-MS/MS method for the identification and quantitation of 110 CCM analytes is presented, where target metabolites are monitored over an 20-min gradient. The analyte panel constitutes amino acids, vitamins, organic acids, nucleic acids, carbohydrates, and lipids. The method employs isotopically labeled standards to enable specific and accurate relative quantitation of CCM compounds based on physicochemical properties and retention time. Quantitation is performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. The method demonstrates strong linearity with an R2 of ≥0.99 with three orders of linear dynamic range and inter-day and intra-day precision with a%CV of <10% for spiked-in quality control samples. We also present three case studies to demonstrate method applicability in the bioprocessing space for developing vaccines and biologics.

An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices.

Sensitive, multiplexed protein quantification remains challenging despite recent advancements in LC-MS assays for targeted protein biomarker quantification. High-sensitivity protein biomarker measurements usually require immuno-affinity enrichment of target protein; a process which is highly dependent on capture reagent and limited in capability to measure multiple analytes. Herein, we report a novel antibody-free platform, which measures multiple biomarkers from complex matrices employing a strategically optimized solid-phase extraction cleanup and orthogonal multidimensional LC-MS. Eight human protein biomarkers with different specifications were spiked into canine plasma as a model investigation system. The developed strategy achieved the desired sensitivity, robustness, and throughput via the following steps: (1) post digestion mixed-mode cation exchange-reverse phase SPE enrichment cleaned up the sample initially; (2) rapid, high-pH peptide fractionation further eliminated background components efficiently while selectively enriched signature peptides (SP) to provide sufficient sensitivity for multiple targets; and (3) trapping-micro-LC-MS analysis delivered high sensitivity comparable to a nano-LC-MS method but with much better robustness and throughput for the final analysis. Compared with a conventional LC-MS assay with direct protein digestion and limited clean-up, analysis with this antibody-free platform improved the LLOQ by 1-2 orders of magnitude for the eight protein biomarkers, reaching as low as 5 ng/mL in plasma, with feasible robustness and throughput. This platform was applied for the quantification of biomarkers of respiratory conditions in patients with various lung diseases, demonstrating real-world applicability.

Polysaccharide identification through oligosaccharide fingerprinting.

The identification of polysaccharide structures in complex samples remains a unique challenge complicated by the lack of specific tools for polymeric mixtures. In this work, we present a method that depolymerizes polysaccharides to generate diagnostic oligosaccharide markers that are then analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS). Rapid identification of food polysaccharides was performed by aligning the identified oligosaccharides with a library of oligosaccharide markers generated from standard polysaccharides. Measurements of standard and food polysaccharides were performed to obtain the contributions of the identified polysaccharides using percent peak coverage and angle cosine methods. The method was validated using a synthetic mixture of standard polysaccharides while the reproducibility was confirmed with experimental triplicates of butternut squash samples, where standard deviation was less than 3% for the relative abundance of oligosaccharides. The method was further employed to examine diverse set of food samples.

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.

Function without Structures: The Need for In-Depth Analysis of Dietary Carbohydrates.

Carbohydrates make up the largest component of plant-based foods and have long been known to provide fuel. However, many carbohydrates possess intrinsic biological activities that are dictated by their structures. Carbohydrates are the most abundant biopolymers in nature and are also the most structurally complicated and diverse. Consequently, the structural analysis of carbohydrates remains severely limited. To further understand their biological activities, we need new analytical tools to analyze the different classes of carbohydrates that range in size from monosaccharides to polysaccharides. These tools must be capable of rapid throughput with highly sensitive quantitation for use in clinical studies that probe their fate in human and animal fluids and tissues.