Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

Transcription

1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 9451 Weinheim, 28

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 9451 Weinheim, 28 Supporting Information for A Versatile Gold Surface Approach for Fabrication and Interrogation of Glycoarrays Zheng-Liang Zhi, Nicolas Laurent, Andrew K. Powell, Rositsa Karamanska, Margherita Fais, Josef Voglmeir, Adam Wright, Jonathan M. Blackburn, Paul R. Crocker, David A. Russell, Sabine Flitsch, Rob A. Field, and Jeremy E. Turnbull*

3 Contact angle measurements: We have observed that under comparable hydrazide coverage conditions, C17 thiol surface showed much better surface wettability (lower contact angle) than C11 thiol surface (Figure S1A). In fact, the bare gold surface used in our microarray construction has a very poor wettability as indicated by the measured contact angle of over 5. The formation of a monolayer significantly improved the wettability of the surfaces, as expected from the structure of the molecule. Notably, a C17 thiol, either in diluted or undiluted forms, resulted in a better wettable surface compared to that of C11 thiol, indicating that a better packed monolayer and likely a higher density of the reactive groups on the monolayer has been achieved with the C17 thiol linker. Both good wettability and high hydrazide surface density are required for efficient binding, to enhance signal levels and increase detection sensitivity on the gold chip microarrays. QCM experiments: The monolayer-covered surfaces were characterized using QCM which detects the mass deposition on a resonance crystal as a function of change of resonance frequency. Figure S1B shows the frequency change on different monolayer-covered gold-coated resonators as a result of protein adsorption. The data showed that a 1% hydrazide surface coverage monolayer results in a higher nonspecific signal, and a or 8% surface coverage monolayer is more suitable for the study of interactions between immobilized sugars and proteins. Experimental Section Quartz crystal microbalance measurements: QCM measurements were carried out by using a Q-Sense D device equipped with a flow cell (Q-Sense AB, Vastra Frolundra, Sweden). Only one side of the crystal was in contact with the solution in the cell well. A standard gold-coated 5 MHz AT-cut quartz crystal was excited at its second overtone (~25 MHz) and the change in the resonance frequency (F) was recorded. The QCM electrodes were first cleaned with piranha solution, then the electrode was covered with a solution (.1 mg/ml, 1 µl in 5% methanol) of hydrazide-modified C17 thiol linker and incubated at RT for 24 h. Following incubation, the electrode was washed with ethanol and milliq water. Sugars ( µm, 1 µl in 1 M betaine) were then attached onto the QCM electrodes for 1 h. Changes in frequency as a result of injection of a protein (4 µl, 1 nm FGF2) were recorded for each adsorption step. The frequency shifts reported in this paper are the difference between two stable values (Figure S1C).

4 A) B) Contact Angle (deg) %, -hydrazide 1%, -hydrazide %, -COOH 1%, -COOH 1%, -OH Bare gold Frequency shift (Hz) FGF2 with heparin FGF2 without heparin C17 C11 Bare gold Surface 8 1 Hydrazide coverage (%) C) Frequency change (Hz) without sugar with sugar Time (s) Figure S1. A). Surface wettability (evaluated via contact angle measurement with water) of C11 and C17 thiol monolayer-covered and bare gold surfaces. Error bars represent the standard derivation of three independent measurements. Monolayers with terminal groups of hydrazide, carboxylic acid and hydroxyl were compared. Note that there is a significant difference (-5 degrees) between C11 and C17 surfaces for differently-terminated groups. The -OH terminated thiol contains a tri(ethylene glycol) group, whereas -COOH and hydrazide terminated thiols contain hexa(ethylene glycol) groups. Only hydrazide-terminated monolayers were used for glycoarray generation. The contact angles were measured using a home-made optic setup by pipeting a drop of 1 µl ultrapure water onto the gold surfaces at room temperature and ambient humidity. Advancing angles are reported. B). Effect of hydrazide surface coverage of the monolayer on protein binding to immobilized heparin using QCM characterization. The measurements were taken in a single sensor format using stopped-flow mode. Error bars represent the errors of two independent measurements. Heparin (dp-1): µm (.1 mg/ml, in 1 M betaine), 1 µl; FGF2: 1 nm. Surfaces with sugar attachment show specific protein binding. Surfaces without sugar attachment show nonspecific binding. C). Representative recordings of the QCM measurements on the C17-EG- hydrazide monolayers (8% surface coverage) on gold-coated quatz crystal. Heparin (dp- 1): µm (.1 mg/ml, in 1 M betaine), 1 µl for immobilization; FGF2: 1 nm,.4 ml for binding tests.

5 Table S1. Details of the sugars studied No Sugar Structure LSTa (sialyllacto-ntetraose a) LSTb (sialyllacto-ntetraose b) LSTc (sialyllacto-ntetraose c) '-sialyl-nacetyllactosamine '-sialyl-nacetyllactosamine Galß1-GlcNAcß1-Galß1-4Glc Neu5Aca2 Galß1-GlcNAcß1-Galß1-4Glc Neu5Aca2 Galß1-4GlcNAcß1-Galß1-4Glc Neu5Aca2 Neu5Aca 2-Galß1-4GlcNAc Neu5Aca 2-Galß1-4GlcNAc Oligomannose-5 Mana1 \ Mana1 \ Manß1-4GlcNAcß1-4GlcNAc Mana1 / / Mana1 7 a 1--mannobiose Mana 1 \ Manß1-4GlcNAcß1-4GlcNAc 8 a 1--mannobiose Manß1-4GlcNAcß1-4GlcNAc / Mana1 9 Heparin decasaccharide [IdoA(2S)-GlcNS(S)] 5 Note: N-acetylneuraminic acid (NeuNAc, Neu5Ac) is the most common member of the group of sialic acid. The structure shown for heparin is the predominant disaccharide repeat unit.

6 Table S2. MALDI-ToF MS analysis of glycans immobilized on a gold surface glycoarray and detection of enzymatic transformation. Spot Sugar Detected mass [a] Mass after GalT treatment [a,b] 1 β-d-glc β-d-gal α-d-man β-d-glcnac β-d-xyl α-d-xyl α-d-glc(1,4)glc β-d-glcnac(1,4)glc β-d-gal(1,4)glc [a] m/z ratios of the sodium adduct of the mixed disulfide formed by the sugar-terminated alkanethiol and the tri(ethyleneglycol) alkanethiol. [b] mass after incubation of immobilized glycan with β1,4-galt / UDP-Gal.

7 A) Fluorescence intensity WGA Sugar: Sugar concentration (mm) B) Fluorescence intensity C) Con A, three-layer assay Man5 Man2 (1,) Man2 (1,) Heparin 5 1 Sugar concentration (mm) Con A, one-layer assay Fluorescence intensity 4 2 Man5 Man2 (1,) 5 1 Sugar concentration (mm) Figure S2. Bar-graphs of the data for an array of nine sugars binding to WGA (A), and selected sugars binding to Con A (B and C). Data from Figure ; average of five spots ± SD.

8 A) B) Fluorescence intensity µm x1 4 2x1 4 1x1 4 dp-4 dp- dp-8 dp-1 dp-4 dp- dp-8 dp Sugar concentration (µm, in disaccharide) Figure S: Binding of FGF2 to glycoarrays displaying different sized heparin saccharides: A) microarray images; B) data analysis (n=, error bars represent standard derivation of the intensities from three spots). FGF2, 1 nm. Whilst smaller saccharides will bind to FGF2, binding is significantly enhanced in the case of octa- and deca-saccharides, in agreement with previous observations (M. Delehedde, M. Lyon, J.T. Gallagher, P.S. Rudland, D.G. Fernig. Biochem. J. 22,, ).

9 Spec #1=>TR[BP = 1257., 494] % Intensity Figure S4: MALDI-ToF MS analysis of immobilized carbohydrates from a x5 glycoarray. Representative MALDI-ToF MS data is shown for the complete set of sugars arrayed in Figure 5, with masses summarised in Table S1.