On‐Chip Synthesis and Label‐Free Assays of Oligosaccharide Arrays

Abstract

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...

SPOT Synthesis of Peptide Arrays on Self‐Assembled Monolayers and their Evaluation as Enzyme Substrates

“…Although proper quantification is the most challenging issue when dealing with mass spectrometry, some semiquantitative data were obtained by comparing the ratios of the signals of the glycopeptide product and peptide substrate. A similar approach has recently been described [10,27] for obtaining relative yields of reaction. As each set of peptide/glycopeptide differs only by a GalNAc moiety, relative intensities can be compared by using the formula r = SI (x) /(SI (x) +SI (y) ), where r is the relative intensity, SI (x) the sum of the intensities of the glycopeptide signals and SI (y) the sum of the intensities of the parent peptide substrate (all proton, sodium and potassium adducts of alkanethiol and disulfide species are taken into account when detected).…”

“…[2] SAMs present well-defined biocompatible surfaces that are easy to prepare and are amenable to detailed physicochemical analysis of molecular structure and binding interactions, in particular through MALDI-ToF MS [3,4,5] (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) and SPR [6,7,8] (surface plasmon resonance). So far, a broad range of chemical and biochemical reactions have been described [5,9] that are compatible with SAMs, but in all cases the number of consecutive reaction steps conducted on the surface has been small, [10,11] in particular in array format. This raises the question of whether the platform is robust enough to withstand repeated reaction and washing cycles for long biopolymer synthesis.…”

“…The mannosyl residue could then be successfully O-deacetylated by using a solution of sodium methoxide in methanol. [10] The use of glycosyl amino acids with unprotected sugar side chains [29] was also explored for the synthesis of a 12-mer glycopeptide GTTASN-A C H T U N G T R E N N U N G (bGlcNAc)YGTGFA, although the less reactive GlcNAcAsn [29d] required a longer reaction time (2 h). Furthermore, by incubating the surface-bound peptide with the bovine enzyme b1,4-GalT in the presence of UDP-Gal and MnCl 2 , overnight, at 37 8C, enzymatic elongation of the carbohydrate moiety was achieved and yielded the corresponding LacNAc-glycopeptide (Figure 1 A and Figure 4 B, m/z 2197 and 2616).…”

Detection of enzyme-catalyzed polysaccharide synthesis on surfaces

“…Such systems are typically based on mass spectrometry (Ban & Mrksich 2008;Laurent et al 2008b;Zhi et al 2008), radiolabels (Matsuo et al 1992;Donovan et al 1999;Shipp et al 2008), lectin affi nity (Bryan et al 2004;Houseman & Mrksich 2002;Plath et al 2006;, antibody detection (Yu et al 1995), fl uorescence affi nity systems (Blixt et al 2008) and bead-based systems (Halling et al 2005). In some applications where there is a small change in mass after enzymatic conversion, such methods may be the ones of choice.…”

Glycan Microarrays for Decoding the Glycome

“…Subsequently, a number of groups have used microarray technology to assess the specificity of glycosyltransferases. Both the Mrksich (152) and Park (153) groups examined the specificity of bovine β1,4 GalT with libraries of ~20 unique structures. In the former case, the array was constructed on SAMs, allowing quantitative analysis of the products by on-chip MS.…”