ESI Multi-sprayer (MUX) Technology; Sample Throughput; Protein Consumption
For high throughput screening, Schnier reported assaying in parallel eight spin column eluates injected into eight ballistic gradient HPLC systems, which fed into an ESI mass spectrometer equipped with an eight-channel multisprayer system . The cycle time for assaying eight wells in parallel, containing 640 compounds was 2 min, with injections in the overlay mode. Using this procedure, a GPC spin column kinase receptor ESI-MS assay was performed on 25 000 compounds, pooled twice, in 2.6 h. A total of 320 overlapping hits were observed between the two sample pools. It should be pointed out that, when using multisprayer sources, the achieved sensitivity for the mass spectrometer for each sample is approximately reduced by a factor equivalent to the number of sprayers. This can impact the number of hits observed because weak binders may not be detectable due to the sensitivity lost by the use of multiple sprayers.
Table 2.2 lists the number of samples that can be assayed per day using the multiple sprayer technology, assuming 2 min and 10 min per assay with four and eight sprayers. The numbers of samples assayed are quite impressive especially considering the amount of protein consumed per compound. Of course it should be pointed out that, by pooling four or eight GPC spin column eluates, the same efficiencies can be achieved as for the multisprayer systems; however, it is only necessary to use a single sprayer and not suffer the sensitivity losses as with the multisprayer system. It is also informative to compare sample throughput and protein consumption between the GPC spin column/ESI-MS technology with that of high throughput screening (HTS), the standard method used in exploratory pharmaceutical drug screening. As indicated in Table 2.2, the number of compounds assayed per day by GPC spin column/ESI-MS as mixtures of 80 or 400 compounds is equal to or exceeds the numbers assayed by HTS of single compounds, while the amount of protein consumed by GPC spin column methodology greatly exceeds (@15 times) the amount used by HTS. Note, however, that the tandem chromatographic method GPC reversed-phase (RP) HPLC with ESI-MS detection with mixtures of 3750 compounds greatly exceeds the numbers of compounds assayed per day by HTS and is comparable with the amount of protein consumed per compound by HTS.
Table 2.2 GPC spin column/ESI-MS drug screening high throughput aspects: compounds assayed per day and protein consumed per compound.
# Compounds Analyzed/Day
Protein Consumed/ Compound
Single Mixture Sample of 4 Wells/ 4-Way MUX
Mixture of8 Wells/ 8-Way MUX
pmole/ compound mg/compound (MW Protein 25 kDA)
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