Glycan Arrays with Direct MS Readout

Immobilization of fluorous tagged molecules onto a fluorous surface has been conducted by several groups for the formation of microarrays.  Generally the surface has been a fluorous modified glass slide.  Solutions containing the fluorous tagged probe are spotted on the glass slide and the probes are then incubated with a protein, enzyme, or cell lysate.  Visualization of these interactions are usually through fluorescence, either by using a fluorescence tagged protein, a fluorogenic probe, or a fluorescence tagged antibody.  Fluorescence works well with fluorous arrays due to high signal to noise (S/N) ratios and low background fluorescence.

(Sidebar:  As always “fluorescence” and “fluorous” are completely different things related only by etymology.  For an explanation, please see this early F-Blog post)

One thing that fluorescence doesn’t give you, however, is actual structural information.  To get that you need to get that probe off the array and to be able to analyze it using something like MS. Direct MS readout of probes can be done using several methods. Often, however, this requires high ionization energies which results in fragmentation of the molecule.   MALDI, matrix-assisted laser desorption/ionization, lowers the required energy needed to effect ionization, but the addition of matrix often interferes with the desired signal, particularly for low molecular weight analytes.

With fluorous immobilization, Suizdak described a fluorous desorption/ionization on silicon (DIOS) method back in 2007 and a nanostructured initiator MS (NIMS) in 2008.  Neither DIOS nor NIMS requires a low-molecular weight matrix.  Now Prof. Chi-Huey Wong, who was also an author on the NIMS work, introduces another method for making fluorous arrays which use direct MS readout for detection in a recent J. Am. Chem. Soc article.  In this instance they fluorous modified aluminum oxide glass (ACG) slides.  The aluminum oxide coating was rendered fluorous by the attachment of a fluorous phosphonic acid.  Fluorous tagged glycans were then immobilized onto the slide and incubated with cellulases to determine selectivity and activity of the cellulases to the various probe glycans.  The authors note that cellulase activity is particularly interesting because of their potential application to biofuel production.  (Gotta breakdown that cellulose someway!)  

The researchers first prepared the fluorous ACG slides and spotted three fluorous tagged sugars; lactose, Globo H, and Gb5.  After drying they were washed and subjected to MS-TOF analysis.  A very clean, non-fragmented MS signal with high S/N was observed using low laser energy rates.  They also found that the immobilized sugars retained their protein-binding activities by incubating the slides with the appropriate antibodies.  Quite nice actually, since one can use either fluorescence or MS based readout.

With confirmation of the fluorous ACG immobilization they then made some simple arrays of fluorous tagged sugars and incubated them with various cellulases.  As controls they also tested the reactivity and selectivity of the sugars in solution.  They found that selectivities did not alter appreciably, but that reaction rates were slower.  They also found that they could easily identify products from the enzymatic hydrolysis and differentiate various types of cellulase activity.

This paper adds to the growing body of knowledge which validates the use of fluorous techniques in glycomics, microarray, and MS applications.

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