Protein precipitation discussion

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. 

Among the fractionation techniques that have been used to study the speciation of trace element containing species include liquid chromatography, gas chromatography, ultrafiltration, dialysis, protein precipitation, electrophoresis and others. In the following sections the application of the above techniques will be discussed. 

The use of methanol and ethanol, two solvents that can be well mixed with water, should be discussed separately, as they are used for several types of sample preparation on the basis of similar expected effects, but for different purposes. The feature common to both solvents is the observation that most of the sample proteins precipitate when the concentration of these compounds exceeds ca. 40 percent (v/v), thus enabling the analyst to separate the protein fraction by centrifugation or microfiltration. This way, either the proteins not intended for further analysis (e.g., enzymes or proteins that were inadequately hydrolyzed) can be removed [62], or, the purification of Se-containing proteins by successive solvent extractions can be achieved [12]. At concentrations of less than 40 percent (v/v), both methanol and ethanol are usually mixed with 0.1 moll-1 HC1 however, the use of these mixtures entails a relatively low extraction efficiency of Se (10-14 percent). Therefore, they are intended only for the extraction of water-soluble Se species, generally free selenoamino acids [15, 21, 63, 64]. 

The influence of electrolytes divides into two areas, i.e. the influence on nonionic (uncharged) surfactants and that on ionic materials. For nonionic surfactants, the effects are either salting-out or salting-in , in line with observations from the Hoffmeister series of electrolyte effects on protein precipitation. While there has been much discussion over the past 50 years on the molecular mechanism involved, including many words on the structure of water, Ninham and Yaminsky have recently shown in a landmark paper that the phenomena can be explained by dispersion interactions (194). 

Before proceeding to discuss the relevance and methodology of sample pretreatment in bioanalysis, it is worth noting that in a few limited instances, it is possible to inject untreated body fluids directly onto the analytical HPLC column. This approach is not possible in GC. Direct injection is only feasible for samples (urine or bile, for example) containing high drug concentrations of the analyte(s) and very low protein concentrations. In this case, the matrix is simply diluted in deionized water before injection. This approach is difficult for serum or plasma samples due to the problem of protein precipitation on... 

The following discussion of methods used for sample extraction and clean-up attempts to cover the more commonly used techniques as well as those that appear to offer some potential for the future but are not yet routine. A review of microextraction procedures used in analytical toxicology (Flanagan 2006) is mainly concerned with extraction of analytes from plasma with particular emphasis on LLE and protein precipitation however, miniaturized LLE and SPE are also described as promising ancillary methods. 

The term "euglobulin" has not always been used in this sense frequently it has been used to denote proteins precipitated by 0.33 saturated amme um sulfate solution. The two definitions are far from identical, as will become apparent in the subsequent discusnon. Moreover, some authors have denoted as euglobulins only those proteins precipitated by removal of salt at some particular pH—commonly at or near pH 7. This definition is much more restricted than that adopted here the prothrombin fraction of plasma, for instance, is very insoluble at low ionic strength at pH 6, but is soluble in the complete absence of salt at pH 7. Wh shall consistently term such proteins "euglobulins in the present discussion. 

Two methods, the one based on the alkali lability of RNA and the other on the acid lability of both RNA and DNA, appeared simultaneously in 1945 and have provided the analytical foundation for much of the recent research activity revolving about nucleic acids. The former of these methods is that of Schmidt and Thannhauser (abbreviated here as ST) (91) which depends upon the selective conversion of RNA phosphorus to organic acid-soluble phosphorus (mononucleotides) by alkali after removal of acid-soluble phosphates and of phospholipid phosphorus (16 et ante). The method of Schneider (S) (93) extracts the same lipid-free protein precipitate with hot trichloroacetic or perchloric (95) acid which solubilizes both RNA and DNA the estimation of each in the mixture utilizes specific colorimetric reactions for the (purine) ribose and desoxyribose moieties. These two methods will be discussed in connection with the combined method which follows. 

This precipitation process can be carried out rather cleverly on the surface of a reverse phase. If the protein solution is brought into contact with a reversed phase, and the protein has dispersive groups that allow dispersive interactions with the bonded phase, a layer of protein will be adsorbed onto the surface. This is similar to the adsorption of a long chain alcohol on the surface of a reverse phase according to the Langmuir Adsorption Isotherm which has been discussed in an earlier chapter. Now the surface will be covered by a relatively small amount of protein. If, however, the salt concentration is now increased, then the protein already on the surface acts as deposition or seeding sites for the rest of the protein. Removal of the reverse phase will separate the protein from the bulk matrix and the original protein can be recovered from the reverse phase by a separate procedure. 

In view of the above discussion, it should be clear why the author recommends that the laboratory of Neonatology precipitate proteins with a Somogyi filtrate and then perform the hexokinase procedure on the filtrate. Neither the glucose oxidase i rocedure nor the toluidine method have adequate sensitivity thout the use of special micro-equipment and speciaT techniques. 

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Addition of the protein lysozyme to DC89PC dispersions resulted in the formation of conical tubules in ethanol-water solution.149 The scanning electron micrograph in Figure 5.37 shows that precipitate from this system is primarily composed of these cones, with a smaller number of cylinders. The cones exhibit more pronounced helical ridges on their exteriors than pure lipid cylinders, suggesting that protein selectively associates to the helical defects in a similar manner as the colloidal particles discussed above. 

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