An efficient method is presented to determine precision and accuracy of multichannel liquid-handling systems under conditions near to application. The method consists of gravimetrical determination of accuracy and optical determination of precision based on the dilution of absorbing and fluorescent dye solutions in microplates. Mean delivery volume per well can be determined with precision better than a 0.04% coefficient of variation (CV). Optical signal precision, CV(S), is improved by multiwavelength measurements. Precision of absorbance measurement yields a better resolution than precision of fluorescence measurement (0.3% and 1.5%, respectively), indicating that absorbance measurements should be preferred. From CV(S), an upper bound of the precision of the volumes delivered is derived. Method performance is demonstrated with the dispenser CyBi™-Drop and the pipettor CyBi™-Well using different ejection principles; with commonly used fluids; with 96-, 384-, and 1536-well microplates; and with photometric and fluorometric indicators. Precision of the volumes delivered, as obtained with optimized methods, all plate formats, and both devices, is better than 2% CV with 2 µL set volume and about 1% CV with higher set volumes. (Journal of Biomolecular Screening 2004:726-733)

The turnover of phosphomonoester groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated in human erythrocytes by short-term labelling with [32P]P1. The procedure applied ensured a quantitative extraction of erythrocyte polyphosphoinositides as well as their reliable separation for the determinations of pool sizes and specific radioactivities. The pool sizes of phosphatidylinositol (Ptdlns), PtdIns4P and Ptdlns(4,5)P2 are 25, 11 and 44 nmol/ml of cells respectively. Under steady-state conditions, the phosphorylation fluxes from [y-32P]ATP into PtdIns4P and Ptdlns(4,5)P2 are in the ranges 14-22 and 46-94 nmol -h-1 -ml of cells-' respectively. Only 25-60% of total PtdIns4P and 6-10 O of total PtdIns(4,5)P2 take part in the rapid tracer exchange, i.e. are compartmentalized. In isolated erythrocyte ghosts, the turnover of PtdIns4P approximately corresponds to that in intact erythrocytes, although any compartmentation can be excluded in this preparation. Under the conditions of incubation employed, the tumover of Ptdlns(4,5)P2 is more than one order of magnitude smaller in isolated ghosts than that obtained for intact erythrocytes.

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.

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