Glycosyltransferases (GTs) catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. GTs are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new GTs are both difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with analysis by mass spectrometry to screen putative GTs. A total of 14,280 combinations of GT, acceptor and donor in four buffer conditions were screened and led to the identification and characterization of four new GTs. This work is significant because it provides a label-free method for the rapid functional annotation of putative enzymes.

Oligosaccharides, like proteins and DNA, are ubiquitous biopolymers that mediate essential functions in organisms. [1] Yet, an understanding of the many roles that carbohydrates play is still at an early stage and essentially absent when compared to our knowledge of the functions of proteins and nucleic acids. [2] This contrast reflects the lack of convenient and flexible tools for the synthesis and biochemical analysis of oligosaccharides and their conjugates. The development of biochips-glass slides patterned with an array of hundreds to thousands of biomolecules-has been particularly important to studies in genomics and proteomics, and first became practical with the on-chip synthesis of oligonucleotides and peptides. [3,4] Herein we present a strategy for the on-chip synthesis of oligosaccharide arrays and we demonstrate that combining these arrays with mass spectrometry permits us to perform label-free assays of glycosyltransferase activities. This method represents a significant addition to the molecular strategies now used to discover and investigate the biological functions of oligosaccharides.Since our report of a carbohydrate array five years ago, [5] several examples demonstrating the preparation and application of oligosaccaharide arrays have been reported. [6] Wong, Paulson, and co-workers prepared 200 amino-substituted oligosaccharides and immobilized these molecules on N-hydroxysuccinimidate-functionalized glass slides for studies of the binding of lectins and viruses. [7] Gildersleeve and coworkers prepared arrays by printing conjugates of carbohydrates and bovine serum albumin and glycoproteins onto epoxide-functionalized glass slides and used these arrays to profile multiple lectins.[8] Seeberger and co-workers used solid phase methods to prepare seven sulfhydryl-terminated oligosaccharides and immobilized these reagents onto maleimidefunctionalized glass slides for assaying the interactions between proteins and carbohydrate epitopes. [9] In collaboration with the group of Seeberger we prepared thiol-modified high mannose oligosaccaharides, which were immobilized on self-assembled monolayers, for assays involving lectin binding.[10] The effort required to synthesize the carbohydrate reagents in this and related work is substantial, and limits the size of arrays. Additionally, the need for labeling strategies can make it difficult to perform assays and, further, is not well-suited to the identification of unanticipated activities.Our approach takes advantage of the combination of monolayers with matrix assisted laser desorption-ionization mass spectrometry (in a technique referred to as self-assembled monolayers for matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (SAMDI-TOF MS)) to directly observe the synthetic intermediates at the surface and therefore to optimize the reactions used in the synthesis of the oligosaccharides. [11,12] This method is also useful in performing assays of enzymes that modify the immobilized oligosaccharides and has been applied to nanopart...

Oligosaccharides, like proteins and DNA, are ubiquitous biopolymers that mediate essential functions in organisms. [1] Yet, an understanding of the many roles that carbohydrates play is still at an early stage and essentially absent when compared to our knowledge of the functions of proteins and nucleic acids. [2] This contrast reflects the lack of convenient and flexible tools for the synthesis and biochemical analysis of oligosaccharides and their conjugates. The development of biochips-glass slides patterned with an array of hundreds to thousands of biomolecules-has been particularly important to studies in genomics and proteomics, and first became practical with the on-chip synthesis of oligonucleotides and peptides. [3,4] Herein we present a strategy for the on-chip synthesis of oligosaccharide arrays and we demonstrate that combining these arrays with mass spectrometry permits us to perform label-free assays of glycosyltransferase activities. This method represents a significant addition to the molecular strategies now used to discover and investigate the biological functions of oligosaccharides.Since our report of a carbohydrate array five years ago, [5] several examples demonstrating the preparation and application of oligosaccaharide arrays have been reported. [6] Wong, Paulson, and co-workers prepared 200 amino-substituted oligosaccharides and immobilized these molecules on N-hydroxysuccinimidate-functionalized glass slides for studies of the binding of lectins and viruses. [7] Gildersleeve and coworkers prepared arrays by printing conjugates of carbohydrates and bovine serum albumin and glycoproteins onto epoxide-functionalized glass slides and used these arrays to profile multiple lectins.[8] Seeberger and co-workers used solid phase methods to prepare seven sulfhydryl-terminated oligosaccharides and immobilized these reagents onto maleimidefunctionalized glass slides for assaying the interactions between proteins and carbohydrate epitopes. [9] In collaboration with the group of Seeberger we prepared thiol-modified high mannose oligosaccaharides, which were immobilized on self-assembled monolayers, for assays involving lectin binding.[10] The effort required to synthesize the carbohydrate reagents in this and related work is substantial, and limits the size of arrays. Additionally, the need for labeling strategies can make it difficult to perform assays and, further, is not well-suited to the identification of unanticipated activities.Our approach takes advantage of the combination of monolayers with matrix assisted laser desorption-ionization mass spectrometry (in a technique referred to as self-assembled monolayers for matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (SAMDI-TOF MS)) to directly observe the synthetic intermediates at the surface and therefore to optimize the reactions used in the synthesis of the oligosaccharides. [11,12] This method is also useful in performing assays of enzymes that modify the immobilized oligosaccharides and has been applied to nanopart...

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This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.

The antioxidant capability of oil‐soluble green tea extract (OSGT) in canola oil was compared to common synthetic and plant‐derived ingredients with antioxidant function. Three storage conditions were included—22–25 °C ambient, 60 °C heated and air‐purging at 22–25 °C. The oxidation process was monitored by electron spin resonance spectroscopy for free radicals, peroxide value for primary oxidative byproducts, proton nuclear magnetic resonance for the loss of oxidatively labile protons, aldehydes as secondary oxidative byproducts and antioxidants residues. Except for tocopherols, the use of antioxidant ingredients improved the stability of the oils under all conditions. However, the extent of improvement differed under different storages for the same ingredients. Evaluating antioxidant ingredients and interpretation of data when using accelerated conditions need caution. Antioxidant level change under different storage conditions provide new insights on the mechanism working of antioxidants in bulk oil. OSGT has comparable performance to TBHQ under ambient temperature conditions. In conclusion, evaluating antioxidant ingredients under accelerated conditions needs caution. Oil‐soluble green tea extract has comparable antioxidative performance to TBHQ in bulk vegetable oil systems without affecting organoleptic properties of the oils under ambient temperature regardless of temperature or oxygen levels.