Preparation techniques protein precipitation

Use of Protein Precipitation in Tandem with Other Sample Preparation Techniques... 

In real life, sample preparation techniques are utilized in tandem with HPLC/MS/MS. Due to the high selectivity of LC/MS/MS in the SRM mode, small quantities of impurities or interferences in the purified fractions from sample preparation protocols dealing with biological matrices can be tolerated. This is especially true for protein precipitation, which is widely used because of simplicity and rapidity, when high-throughput turn-around is required. High-throughput HPLC is discussed elsewhere in this book. 

However, PPT cannot be considered a true sample preparation technique because it removes plasma proteins only through the addition of a precipitating solvent and subsequent homogenization and centrifugation. 

Before compounds in biological matrices can be analyzed by LC/MS/MS, the samples must undergo a preparation procedure. There are a variety of techniques available for sample preparation including offline sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction) and on-line sample preparation... 

While the idea of limited sample preparation and direct infusion into the MS are promising, this technique has not yet caught on in many laboratories. Ion suppression continues to be a limitation of this technique. It is also a limitation in other fast sample preparation methods and fast analyses. Protein precipitation is a quick and dirty technique, and many components from the plasma matrix remain. With this direct infusion into the MS, these matrix components are also sprayed into the source with the analytes of interest and can cause ion suppression. Additionally, many samples can contain several analytes, often including internal standards. In many instances, these analytes cause self-suppression when infused together into the source. With a proper sample cleanup such as SPE, either off-line or on-line, this technique may prove to be more useful in the future. 

By taking into account the difficulties connected to the slow solvent evaporation technique, a new procedure for nanoparticle preparation by co-precipitation was set up. This technique does not use chlorinated solvents and energetic mixing, which are both known to cause appreciable protein denaturation[17,18]. 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

An experimental complication is the difficulty in effecting molecular interaction between the components. The usual technique for preparing lipid-protein phases in an aqueous environment is to use components of opposite charge. This in turn means that the lipid should be added to the protein in order to obtain a homogeneous complex since a complex separates when a certain critical hydrophobicity is reached. If the precipitate is prepared in the opposite way, the composition of the complex can vary since initially the protein molecule can take up as many lipid molecules as its net charge, and this number can decrease successively with reduction in available lipid molecules. It is thus not possible to prepare lipid— protein—water mixtures, as in the case of other ternary systems, and to wait for equilibrium. Systems were prepared that consisted of lecithin-cardiolipin (L/CL) mixtures with (a) a hydrophobic protein, insulin, and with (b) a protein with high water solubility, bovine serum albumin (BSA). 

Precipitation with ammonium sulphate is by far the most common of the salt-induced reversible precipitation techniques. There are several reasons for this ammonium sulphate solutions can be prepared at sufficiently high concentrations to precipitate nearly all proteins addition of solid ammonium sulphate produces only a very limited warming of the solution owing to the low enthalpy of solution the density of saturated ammonium sulphate solutions is less than that of protein, so that protein precipitates can be readily collected by centrifugation and finally, concentrated ammonium sulphate solutions are bacteriostatic. 

Certain techniques are very good at particular tasks (such as protein precipitation for removal of proteins and cellular components), but perhaps not as good in general. With the emphasis on greater efficiency, many of these approaches have become automated or semiautomated. Thus, there has been greater emphasis on the 96-well formats an approach that can lend itself more readily to automated workstations. This format has been used successfully for protein precipitation, liquid—liquid extraction, and solid-phase extraction [8-10]. Ultrafiltration (UF) in a 96-well format is also being evaluated and shows some potential, but products and applications are not yet fully developed. Automated techniques for sample preparation and each of the sample preparation techniques listed in Table 1 are described below. 

For this reason, when a cassette is composed of structurally diverse compounds, it is usually prudent to use protein precipitation as a plasma sample preparation technique. Although the absolute recovery will vary from compound to compound, this approach can be applied with fairly universal success across a wide structural range of compounds and assay development time will be minimal. Generic solid-phase extraction protocols can also be effective, but will experience a higher (10 to 15% of compounds) failure rate. Chapter 6 contains examples of generic extraction protocols that can be adapted for use with cassettes containing diverse NCEs. 

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

Protein precipitation is often used as the initial sample-preparation scheme in the analysis of small drug molecules, since one universal procedure is followed for all compounds and method development is unnecessary. The speed of this technique presents a real time savings. The high resolving power of LC-MS/MS analytical methods accommodates this nonselective cleanup procedure. This mode of sample preparation is also inexpensive and does not chemically alter the analyte. Typical sample matrices that are used with protein-precipitation techniques are plasma, serum, tissue homogenates, and in vitro incubation mixtures. In general terms, a sensitivity of 1-10 ng/mL is often achieved from 50- 4L sample volumes with this technique with LC-MS/MS analysis. 

Filtration is used as a sample-preparation method to remove particulates and debris that can potentially foul the LC lines, column frits, or mass spectrometer interface. Also, it is generally accepted that all workstations and pipetting systems can beneht from sample hltration because of the universal issues related to plasma clot formation that introduce pipetting challenges. Applications that use hltration include the removal of a mass of precipitated protein or of debris from raw plasma before use with any of the traditional sample-preparation techniques, as well as direct injection techniques (turbulent how... 

A recent shift toward the discovery and development of new chemical entities that have greater potency has required their dosing at lower levels popular sample-preparation techniques such as protein precipitation are less useful for analyte concentrations below 1 ng/mL. Additionally, the frequent use of mouse plasma necessitates the use of sample volumes <50 pL and effectively miniaturizes the sample-preparation process. In these situations, SPE is especially appealing due to packaging of sorbents within small-diameter, thin disks requiring dramatically lower solvent and elution volumes [54,78-80]. Also, the benefits of disks are often attainable with small sorbent bed masses packed in colunms that are now available in bed masses as low as 2 mg [81]. 

Although many individual choices for sample preparation exist, sometimes the combination of two techniques yields a more desirable result. Within a drug discovery environment, however, throughput and cost are important factors that influence the choice of sample-preparation methods. The protein-precipitation procedure can quickly yield a sample that is ready for analysis, but its potential for carryover of matrix interferences is problematic. The isolated supernatant can be filtered or centrifuged before injection, but these procedures remove only particulates or proteinaceous material, not the materials that cause matrix interferences. Thus, protein precipitation is sometimes followed by LLE, SPE, or an on-line technique for a more selective cleanup before analysis. A particularly challenging sample-preparation requirement is the analysis of animal tissues, as is commonly performed in drug-discovery support laboratories that perform drug uptake studies. Here, a series of sequential sample-preparation steps are common [121]. 

Many different sample preparation techniques are available to the drug discovery scientist. Off-line sample preparation procedures include protein precipitation, filtration, dilution followed by injection, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Typically, these procedures are performed in an automated, high-throughput mode that features a 96-well plate format. Online sample preparation procedures include SPE and turbulent flow chromatography (TFC) with conventional chromatographic media or restricted access media (RAM). These online approaches are often simple and easy to automate. 

Attempts to develop automated methods from first principles require that sufficient time, hiunan skills, and resource are available and that these are not critical elements in the method development equation. Therefore, before beginning any work, it is important that sufficient information be assembled to enable a risk and technical feasibility assessment to be carried out on the proposed automated sample preparation method. Thus, it is important that the method selected comprises sample preparation processes that are amenable to automation. The techniques that have the greatest potential for automation are solid-phase extraction and high-performance liquid chromatography. Other sample preparation techniques such as liquid-liquid extraction, protein precipitation, and ultrafiltration are either difficult to automate or may not be cost-efficient to do so compared with alternative approaches. 

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