Protein precipitation methods for

Jiang, L., He, L. and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography A 1 023, 31 7-320. 

C. J. Kitchen, A. Q. Wang, D. G. Musson, A. Y. Yang, and A. L. Eisher, A semiau-tomated 96-well protein precipitation method for the determination of mon-telukast in human plasma using high performance liquid chromatography/ fluorescence detection, J. Pharm. Biomed. Anal. 31 (2003), 647-654. 

Hagerman, A. E. Butler L. G. Protein precipitation method for the quantitative determination of tannins. J. Agric. Food Chem. 1978,26,809-812. 

As mentioned before, the amount of soluble tannin that causes astringency in persimmon fruits is usually estimated visually by the tannin print method and can be measured quantitatively by the Folin-Denis method. There is also a protein precipitation method for the measurement of soluble tannins (Hagerman and Butler 1978). In that method, the soluble tannin content is assayed by the addition of the sample to a standard solution of protein and the isolation of insoluble tannin-protein complexes. The complexes are dissolved in alkaline solution, to which ferric chloride is added. The absorbance of the solution at 510 nm is measured. 

More information on the comparative evaluation of protein precipitation methods may be obtained from Lei and coworkers.163 An interesting comparison of protein precipitation (PPT) and solid phase extraction (SPE) methods was presented in a technical library publication from Millipore164 that describes use of its Multi-SPE-MPC extraction plate and Multiscreen deep well Solvinert filter plate for SPE and PPT, respectively (Figure 1.45). A Biohit Proline multichannel pipette was used to add 400 /iL of acetonitrile to each well of the filter plate and then, using the pipette s double aspiration program, 100 /iL of spiked serum was aspirated and 100 /iL of acetonitrile from the filter plate was aspirated to initiate protein precipitation in the pipette tip. The mixture was deposited back in the filter plate and shaken vigorously for 2 min. 

Non immunochemical Turbidimetric and Nephelometric Methods. Precipitation of protein for turbidimetric or nephelometric assays is achieved with sulfosahcylic acid alone, or with sulfosahcyfic acid in combination with sodium sulfate or trichloroacetic acid (TCA), or with TCA alone. Precipitation methods for total protein assay depend on formation of a fine precipitate of uniform, insoluble protein particles, which scatter incident light in suspension. 

Each of these approaches is practical for performing high throughput protein-precipitation methods. The determining factors for selection of the collection-plate format over the filtration plate, or vice versa, include the extent of available hardware and automation accommodating the microplate format, total cost of materials, and thus the cost per sample, number of physical manipulations, and the degree of transfer loss deemed acceptable. 

Factor VIII, immunoglobulin, and albumin are all held as protein precipitates, the first as cryoprecipitate and the others as the Cohn fractions FI + II + III (or FII + III) and FIV + V (or FV), respectively (Table 7, Fig. 2). Similarly, Fractions FIVj + FIV can provide an intermediate product for the preparation of antithrombin III and a-1-proteinase inhibitor. This abiUty to reduce plasma to a number of compact, stable, intermediate products, together with the bacteriacidal properties of cold-ethanol, are the principal reasons these methods are stiU used industrially. 

In dye-binding tests, milk is mixed with excess acidic dye solution where the protein binds the dye in a constant ratio and forms a precipitate. After the dye—protein interaction takes place, the mixture is centrifuged and the optical density of the supernatant is determined. Utilization of the dye is thus measured and from it the protein content determined. Several methods for appHcation of dye-binding techniques to milk are given (24,25). 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

Urea Enzymatic Dialysis Method. This method (16) uses 8 M urea [57-13-6] to gelatinize and facUitate removal of starch and promote extraction of the soluble fiber at mild (50°C) temperatures. EoUowing digestion with heat-stable a-amylase and protease, IDE is isolated by filtration or I DE is obtained after ethanol precipitation. Values for I DE are comparable to those obtained by the methods described eadier, and this method is less time-consuming than are the two AO AC-approved methods. Corrections for protein are required as in the AO AC methods. 

Initially fermentation broth has to be characterised on the viscosity of the fluid. If the presence of the biomass or cells causes trouble, they have to be removed. Tire product is stored inside the cells, the cells must be ruptured and the product must be freed. Intracellular protein can easily be precipitated, settled or filtered. In fact the product in diluted broth may not be economical enough for efficient recovery. Enrichment of the product from the bioreactor effluents for increasing product concentration may reduce the cost of product recovery. There are several economical methods for pure product recovery, such as crystallisation of the product from the concentrated broth or liquid phase. Even small amounts of cellular proteins can be lyophilised or dried from crude solution of biological products such as hormone or enzymes.2,3... 

From animal tissue, especially bovine lung and liver (e. g. autolysis of comminuted tissue parts, heating with ammonium sulfate in alkaline solution, filtration and acidification yield heparin as complex with protein, removal of fat with alcohol and treatment with trypsine for the purpose of decomposition of proteins, precipitation with alcohol and various purification methods). 

A method for the determination of formaldehyde in the presence of acetaldehyde was developed by Nicolet and Shinn.88,100 103 After the excess periodate had been destroyed, these workers swept the acetaldehyde (from the neutral reaction mixture) into a sodium bisulfite solution by means of a stream of carbon dioxide. The acetaldehyde was measured by conventional, bisulfite methods, and the residual formaldehyde was precipitated with Dimedon. This procedure was applied to protein hydroly-zates and to terminal deoxy structures of carbohydrates.88,280 ... 

The study concluded that Once wash steps are optimized, samples prepared by solid phase extraction are cleaner than those prepared by protein precipitation. Samples prepared by extraction with a Multi-SPE plate resulted in lower LOQs than samples prepared by solvent precipitation. Drug recoveries were acceptable (>80%) for both the SPE and the solvent precipitation methods. Well-to-well reproducibility of samples was slightly better with extraction with a Multi-SPE plate. Evaporation and reconstitution, while more time-consuming, yield better chromatographic performance, allow analysis of lower concentration samples, and require optimization for good analyte recovery. 

For discovery PK assays, the most common sample preparation procedure is protein precipitation161720 24 because it is fast, easy to automate, and requires no method development. While protein precipitation typically will not provide as clean a sample as will alternative procedures, it is sufficient for most discovery PK samples that use HPLC/MS/MS for the analytical step.21101... 

In some cases, so called direct plasma injection techniques may be used23 83 104 108 instead of protein precipitation for loading plasma samples onto an HPLC/MS/MS system. Some direct plasma injection systems use a column switching technique in which the plasma is loaded onto an extraction column that retains the small molecules. The other plasma components are sent to waste and the flow is switched so that the small molecules are eluted onto an analytical column that connects to the MS/MS.23 83 108 One variation of the column switching method is turbulent flow chromatography commercialized by Cohesive Technologies (now part of Thermo, San Jose, CA).23... 

The most common (off-line) sample preparation procedures after protein precipitation are solid phase extraction and liquid-liquid extraction. Multiple vendors and available chemistries utilize 96-well plates for solid phase extraction systems and liquid-liquid extraction procedures. Both extraction process can prepare samples for HPLC/MS/MS assay. Jemal et al.110 compared liquid-liquid extraction in a 96-well plate to semi-automated solid phase extraction in a 96-well plate for a carboxylic acid containing analyte in a human plasma matrix and reported that both clean-up procedures worked well. Yang et al.111 112 described two validated methods for compounds in plasma using semi-automated 96-well plate solid phase extraction procedures. Zimmer et al.113 compared solid phase extraction and liquid-liquid extraction to a turbulent flow chromatography clean-up for two test compounds in plasma all three clean-up approaches led to HPLC/MS/MS assays that met GLP requirements. 

For discovery PK samples, rapid method development is required. For HPLC/MS/MS assays, method development can be achieved within 2 hr if no unusual problems are encountered. Xu et al.101 described a process for rapid method development as part of the discovery PK paradigm. As shown in Figure 7.4, the systematic process is based on using protein precipitation as the sample clean-up step and generic HPLC conditions for the HPLC/MS/MS assay. 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

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