Qian Wang1; Zachery Lewis2, 3; Andres Guerrero1; David Mills2, 3; Carlito Lebrilla1

1Department of Chemistry, UC Davis, Davis, CA; 2Department of Viticulture & Enology, UC Davis, Davis, CA; 3Department of Food Science & Technology, UC Davis, Davis, CA

Novel Aspect
A fast and simple method employing direct MALDI MS to compare the affinities between oligosaccharides and bacteria.

A necessary step of bacterial colonization and infection is the adherence of the bacteria to host cell surfaces. The adherence factors of bacteria are called adhesins, which consist of two major types: protein adhesins and carbohydrate adhesins. The adherence factors of host cells which adhesins recognize are called receptors, which are usually carbohydrate or peptide residues on the surface of host cells. The specific adherence of adhesins to receptors determines the tissue specificity of binding. In this study, the goal is to develop a rapid mass spectrometry method with the use of MALDI TOF/TOF mass spectrometer to analyze the specific affinities between different bacteria and oligosaccharides which are a major type of receptors.

First, oligosaccharides solutions are applied on the MALDI spots and the MALDI plate (with immobilized phenylboronic acid on the surface which is able to bind to oligosaccharides covalently) is incubated in humidified atmosphere. After that, the unbound oligosaccharides are washed away. Then bacteria suspension is applied on the MALDI spots and the MALDI plate is incubated at 37 oC. The unbound bacteria are washed away after incubation (For the control spots, unbound bacteria are not washed away). Lastly the matrix is applied and mass spectra are obtained. If the mass spectrum matches that of the control spot, then the affinity is approved. If no (or weak) mass spectrum is observed then the affinity is disproved (or the affinity is weak).

Preliminary Data
We studied the amount of oligosaccharides to saturate the MALDI spots. If the spots are not saturated by oligosaccharides, then bacteria may bind to the plate directly which will produce false signal. Different amounts of oligosaccharides were applied on each spot and mass spectra were obtained. Starting from the spot with 10-10 mole oligosaccharides, the peak of sodiated molecular ion of the oligosaccharide was observed suggesting that the spot had been saturated. So 10-10 mole of oligosaccharides is enough to saturate a spot.

The mass spectra of the control spots were obtained. Bifidobacterium longum subsp. infantis ATCC 15697, a commensal bacteria abundant in breast fed infants, Lacto-N-tetraose, disialyllacto-N-tetraose, 3-sialyllactose, 3’-sialyllactose, 6’-sialyllactose and Lewis B tetrasaccharide were used to probe the binding interactions. Bacteria that were bound to the specific oligosaccharide-activated surfaces were analyzed by direct MALDI. The mass spectra from these compounds were the same suggesting that mass spectra were not dependent on the oligosaccharides. Escherichia Coli DH5-alpha was also applied to the surface. These bacteria yielded spectra that were distinct from B. infantis. The mass spectra of B. infantis and E. Coli with the same oligosaccharide are unique and reproducible suggesting that this method is able to differentiate different types of bacteria. An effective washing method to remove unbound bacteria was developed to probe the various bacterial surface and determine the specific affinity of the bacteria to the oligosaccharides.