MALDI-TOF mass spectrometric analysis of enzyme activity and lectin trapping on an array of N-glycans.

Glycan microarrays are an established platform for the highthroughput screening of substrate specificities of carbohydrate-binding proteins and processing enzymes. The most common detection method, which is based on fluorescently tagged lectins, can only give a measure of binding specificity, as quantification is often compromised by the specific lectin affinity. Therefore, a label-free technique that could overcome these analytical problems is needed for the analysis of array spot compositions. MALDI-TOF-based assays on surface-bound carbohydrates have been reported. Mrksich and co-workers have shown that this technique can be used to study enzyme activity or to trap affinity ligands on biofunctionalized selfassembled monolayers (SAMs) of oligoethylene glycols on gold surfaces. Unfortunately, the covalent attachment of glycans to the monolayer requires high ligand concentrations that are not suitable when working with complex oligosaccharides. More recently, Wong, Siuzdak, and co-workers studied 2,3-sialyltransferase and b-galactosidase activity on fluorous-tagged lactose immobilized on a perfluorinated surface. The difficult preparation of the nanostructured surface and the multistep tagging procedure, however, render this approach less appealing for routine and high-throughput use in on-chip mass spectrometric analysis of enzyme activity. Inspired by the flexible and mobile organization of glycolipids in lipid bilayers, we present herein a novel strategy for the surface-based MALDI-TOF analysis of glycan arrays that is conceptually a fusion of the approaches of Wong, Siuzdak, Mrksich, and their respective co-workers. Glycans are functionalized with a lipid tag (Scheme 1) and noncovalently immobilized on the MALDI plate by insertion into a self-assembled alkylthiolate monolayer. This setup was tested in a series of glycomics applications. The facile preparation of both a hydrophobic MALDI plate and tagged ligands, as well as the general applicability for large oligosaccharides make this method stand out from other surface-based MALDI-TOF approaches. Hydrophobic surfaces have been applied to MALDI-TOF-based proteomics for sample desalting, but, to the best of our knowledge, these surfaces have not been applied to the oriented immobilization of lipid-tagged biomolecules for surface MALDI-TOF analysis. We chose a commercially available gold-coated MALDI sample plate as surface to prepare a hydrophobic selfassembling monolayer of 1-undecanethiol by following a published procedure. Analysis of the monolayer under standard MALDI-TOF conditions showed only peaks corresponding to known matrix ions, while no disulfide or thiolate ions were detected (Figure 1). The hydrophobically tagged carbohydrate ligands 1–10 used in this study were prepared from synthetic N-glycan structures (2–8) by conjugation with stearic acid or from commercial reducing sugars by reductive amination (1, 9, and 10). Glycan arrays (see the Supporting Information) were made by spotting the conjugates 2–10 onto individual wells of the hydrophobic sample plate, drying, and rinsing the plates with water to remove unbound material. MALDI-TOF analysis using 2,4,6trihydroxy-acetyophenone (THAP) as matrix showed strong ion signal intensities with signal-to-noise values of typically 100 or higher for all compounds; these values are comparable to those reported by Siuzdak and co-workers. Ions were detected as sodium adducts with a detection limit of around 5 picomol. The maximum surface capacity for glycan immobilization was determined by deposition of increasing amounts of conjugate 2, washing, and measurement of the signal for 2 normalized to an internal standard. Surface saturation was reached after deposition of around 2 nmol of conjugate 2 per well, which translates to a surface concentration of 0.2 mmolmm . To avoid the unnecessary waste of valuable analytes, glycans were spotted at half the saturation concentration without compromising signal intensity. The stability of the immobilized glycans to repeated washing with aqueous buffers, water, or organic solvents was then determined. Tagged glycans typically resisted 3–5 wash cycles of 1 minute duration without significant reduction of signal intensity, and the intensity of the model glycan 2 was essentially unchanged after 60 seconds of continuous sonication (Figure 1d). However, the glycans were completely removed from the surface [*] Dr. A. Sanchez-Ruiz, Dr. S. Serna, Dr. N. Ruiz, Prof. Dr. M. Martin-Lomas, Dr. N. -C. Reichardt Biofunctional Nanomaterials Unit, CICbiomaGUNE Paseo Miramon 182, 20009 San Sebastian (Spain) Fax: (+ 34)943-005-314 E-mail: nreichardt@cicbiomagune.es