Trent Northen and co-workers have been using fluorous immobilization in conjunction with nanostructure initiated mass spec (NIMS) in a number of different applications. Dr. Northen was one of the inventors of NIMS while working in Gary Siuzdak’s labs at The Scripps Research Institute. NIMS uses a fluorous siloxane as a initiator and is analogous to MALDI. Nimzyme is what they dubbed the process by which a fluorous tagged substrate is immobilized on the fluorous siloxane, undergoes a reaction on the surface, and the products then analyzed by MS. In all the previous cases that have been published the probe molecules have all been tagged with the same fluorous tag, so different substrates lead to different products that are recognized by their mass differences.
But what do you do if your different substrates all lead to the same product? How then do you differentiate between them? You use a different fluorous tag for each substrate, of course. Now the mass differentiation comes not from the substrate, but from the fluorous tag which encodes for each substrate and that’s exactly what Northen et al have done in their latest publication in Rapid Communications in Mass Spectrometry. They encoded three different disaccharides, maltose (S1), lactose (S2), and cellobiose (S3) with a unique, and mass differentiated, fluorous tag as seen below. Note that the mass (427.2) of the disaccharide portion is the same for each substrate and that they only differ in stereochemistry, but that after tagging each has a unique mass. Without the fluorous tag there would not be a simple way to differentiate between these species by MS.
The authors took their mixture of substrates and incubated it with three different enzymes and a mixture of all three. An aliquot of the reaction was then spotted on a NIMS surface with the fluorous tagged compounds being retained. As expected, each enzyme reacted stereospecifically with only one of the substrates; a substrate that was easily identified by its unique mass courtesy of the fluorous tag. When using the mixture of the enzymes, all three were hydrolyzed to a uniquely fluorous tagged glucose. The authors went on to demonstrate the use of cell lysates rather than purified enzymes and found essentially the same results. The fluorous NIMS surface is critical in this since the fluorous tagged substrates and products and retained on the surface once spotted and an on-surface enrichment conducted.
The use of different fluorous tags for the encoding of substrates is the key element in fluorous mixture synthesis (FMS), where mixtures of substrates can be prepared at once and later demixed using fluorous HPLC (F-HPLC) based on fluorous tag length. In this work, the application is not synthetic, but analytical, and the compounds can be unequivocally indentified through mass, even when working with mixtures of substrates and enzymes. As the authors note given the vast array of combinations of fluorous tags and linkers that could be made, each with distinct masses, this could be a general method by which to encode enzyme substrate libraries.