Protein precipitation screening

Table 4 Design Table for Protein Precipitation Screening Study (2 Full Factorial Experimental Design with Center Point)...

A regularly formed crystal of reasonable size (typically >500 pm in each dimension) is required for X-ray diffraction. Samples of pure protein are screened against a matrix of buffers, additives, or precipitants for conditions under which they form crystals. This can require many thousands of trials and has benefited from increased automation over the past five years. Most large crystallographic laboratories now have robotics systems, and the most sophisticated also automate the visualization of the crystallization experiments, to monitor the appearance of crystalline material. Such developments [e.g., Ref. 1] are adding computer visualization and pattern recognition to the informatics requirements. 

Protein Precipitation Techniques and Instrumentation for High-Throughput Screening... 

Once a hit has been found, the next step is to conduct a finer screen around these conditions by varying the concentrations of protein, precipitant, addition of additives, etc. In most cases, more than three different ingredients may be needed in each trial, making it difficult to manually pipette all the ingredients into one drop directly under the oil. Thus the procedure in Protocol 3.2 should be followed. 

Among the possible limitations for the use of NMR as a screening tool, the relative insensitivity of NMR as compared with other spectroscopical methods is the more important. This fact imposes the use of high total compound concentration for the ligand molecules and limits the mixture size that can be tested because of compound solubility, protein precipitation and non-specific binding. We shall see in several examples that this limitation turns out to be inherently less important as the use of new probes and higher fields become available, lowering the threshold of sample concentration to be used in these studies. 

An example for an automated stability test in plasma is described by Linget and du Vignaud (1999). Incubations are performed on a 215 Gilson liquid handler. Incubation was done at substrate concentrations of 50 pM on 96 deep well plates. Each incubation tube contained 375 pL of a 200 pM test compound solution (in 0.1 M Tris buffer with 3% BSA, added to assist dissolution of compounds with poor solubility) and 1125 pL of plasma. Samples are taken after incubation times of 0, 1, 2, 3, 4 and 5 min. At each of these time points an aliquot of the incubation mixture was transferred from the incubation tube into a well in a 96 deep well plate containing an equal volume of acetonitrile for quenching by protein precipitation followed by centrifugation of the plates. Supernatants were analyzed by HPLC for metabolic screening. 

The post-colunm infusion setup has been widely applied in matrix effects studies, e.g., in developing methods for the quantitative screening of drag discovery compoimds by fast-gradient (1 min ran time) LC-MS [83-85], in the optimization of the sample pretreatment in the analysis of methadone, comparing four off-hne and three on-hne sample pretreatment methods [77], and in evaluating various protein precipitating additives [86],... 

Baseline separation was achieved between the candidate drug and the two metabohtes after extraction of the compounds from human plasma following protein precipitation. The LOQ was improved by a factor of 5 for the HPLC-MS/MS method after chemometric optimization. The UPLC-MS/MS method resulted in much lower operational cost, and therefore it was decided to optimize that method. Significant factors from the experimental screening were optimized via central com-... 

For biological fluids, SPE has been widely employed for the extraction of doping agents and veterinary drugs from urine and plasma. However, a number of authors have reported the use of simple sample treatments such as plasma protein precipitation or urine dilute-and-shoot (64,65). These procedures were only used during the initial screening step and not the confirmation step. In addition, TOF/MS devices were generally employed to compensate for the lack of selectivity and sensitivity afforded by these sample preparation procedures. 

The liquid-liquid free interface diffusion (FID) method, in which protein and precipitant solutions are carefully superimposed and left to slowly mix diffusively, was least used in the past due to handling difficulties. However, in the last 4 years the free interface technique has experienced a revival for both screening and optimization procedures. The... 

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