Spin Column Media: Advantages and Disadvantages, Volatile vs Non-volatile Buffers
The most popular GPC gel media are polyacrylamides and sephadexes (see Table 2.1). When used in the spin column format, compounds with MWs within the fractionation range of the gels generally are retained in the pores of the gel beads and compounds with MWs greater than that of the fractionation range generally pass through the columns unrestricted. Since most protein and protein-drug complexes in pharmaceutical screening programs have MWs greater than 15 000 Da and the small molecules have MWs less than 1000 Da, the gel media used most often are the polyacrylamides P2, P4, P6 and the sephadexes G10, G15, G25. Highest sensitivity for identifying non-covalently bound small molecules to protein can occur using the GPC spin column/ESI-MS methodology when in control experiments the maximum amount of protein passes through the spin column. Likewise, in control experiments, all the small molecules should be retained on the column. In general, as the upper mass limit of the fractionation range for the gel media decreases, the amount of protein transmitted through the spin column increases with a greater risk for the transmission of the small molecule through the column. In screening studies with the insulin-like growth factor receptor (IGFr) protein (predicted MW 35 065 Da), the transmission of protein through identical spin column volumes for P6, G25 and P2 were 5%, 17% and 34%, respectively, relative to the response of the same amount of protein directly analyzed by ESI-MS. Clearly, P2 was the preferred gel since protein transmission was the highest and in control experiments none of the small molecules evaluated gave false positive results.
The gel media should be hydrated and washed with a buffer system compatible with maintaining the protein target in its native state. To thoroughly remove contaminants present in the gels, multiple washes are necessary. Sephadex beads generally require more numerous washes than polyacrylamide beads. It is preferable to wash the columns with volatile buffers (ammonium acetate, ammonium bicarbonate systems) rather than non-volatile buffers (phosphate, sulfate systems) since they are more compatible with the ESI-MS assay and lead to minimal ion suppression. The gel columns are prepared by loading the washed gel into the column and centrifuging the column at 775 g for 3 min to gently remove excess buffer without dehydrating or drying the gel. This whole procedure of gel hydration, washing and column preparation can also be performed after loading dry gel into the miniature column and is the preferred method for preparing gel columns with 96-well microtiter plates. A useful spin column feature is the ability to exchange non-volatile buffers, often present in proteins, with the volatile buffers present in the spin column. If volatile buffers are present in the spin column eluate, flow injection ESI-MS is possible but if not HPLC ESI-MS is required.
All the GPC spin columns described above are inexpensive, disposable and designed for single use, avoiding all possibilities for cross contamination of samples. A very desirable feature of the GPC spin column technique is that the eluate contains principally only positive hits of non-covalently bound ligands and all other small molecules are absent, unlike other screening techniques, e.g., centrifugal ultrafiltration (see Chapter 4), where the tight binders are enriched while still retaining chemical noise from unenriched components of the mixture. On rare occasions, false positive results are obtained when a small molecule passes through the GPC spin column unfractionated, together with the protein and not as a non-covalently bound complex. This can be easily verified by assaying the small molecule alone in the absence of protein via the GPC spin column/ESI-MS methodology. From screening studies of large libraries, more false positive results were observed with sephadex beads than with polyacrylamide beads. The most common false positive results were obtained with organic molecules containing poly-carboxylic acid, poly-cyano, sulfate, poly-sulfate, phosphate and polyphosphate moieties. Most of these compounds are not likely drug candidates.
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