Protein sample preparation

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

Sample preparation, protein analysis, 78 Saponification of lipids, 438-439... 

For sample preparation, protein precipitation or liq-uid/liquid extraction can also be applied instead of solid phase extraction. Gluth et al. (1988) described for a toxicokinetic assay for Sotalol a threefold combination of these principles. A protein precipitation using 5 M perchloric acid was followed by a liquid/liquid extraction into a mixture of n-pentanol-chloroform 1/3 at pH 9. Thereafter, the organic phase was transferred to another glass tube and the analyte back extracted into 0.05 M sulfuric acid. 

For sample preparation, protein precipitation or solid phase extraction or liquid/liquid extraction or any... 

After sample preparation, protein yield can be measured by optical density. Several methods are... 

Pharmacoproteomics Proteome analysis comprises three sequential steps sample preparation, protein separation and mapping, and protein characterization. 

Applicable only for soluble chitin/chitosan samples requires sample preparation proteins, organic pigments and humidity are interferences applicable for a limited range of the DA, where the sample is soluble in the solvent. 

Although there is an increasing trend toward precolumn derivatization methods, there are several advantages to an ion-exchange separation with postcolumn derivatization. The first is that there is little sample preparation protein hydrolysates can be injected directly into the column for analysis by LC. Unlike precolumn methods, it is not necessary to separate reaction side-products from the reaction mixture instead, they become a source of background. With postcolumn methods there is more sample-to-sample consistency because of the robustness of the ion-exchange separation and the fact that the reaction time is determined by the column size. 

The presence of proteins in biological fluids constitutes an interference in many analyses and their removal is often the major part of sample preparation. Proteins are typically removed by precipitation with acetonitrile or sulfosalicylic acid. Ultrafiltration or ultracentrifugation can also be used. 

Here, we will describe a range of applications of MALDI-MS, from the concepts of in-depth analysis of purified proteins to applications of MALDI-MS in a broader, proteomics-based research where proteins are identified, characterized, and quantified. In addihon, issues of sample preparation, protein characterization and identification strategies and bioinformatic tools for data interpretation wiU be discussed. The concepts of peptide fragmentation, sequencing and derivatization, analysis of post-translational modifications and the clinical apphcations of MALDI-MS are also briefly outlined. 

Considering the numerous applications, heart-cut LC-LC has convincingly proven its value. Nevertheless, in LC-LC specific method development is generally needed for each analyte. Moreover, heart-cut procedures require accurate timing and, therefore, the performance of the first analytical column in particular should be highly stable to thus yield reproducible retention times. This often means that in LC-LC some kind of sample preparation remains necessary (see Table 11.1) in order to protect the first column from proteins and particulate matter, and to guarantee its lifetime. 

Liquid samples might appear to be easier to prepare for LC analysis than solids, particularly if the compounds of interest are present in high concentration. In some cases this may be true and the first example given below requires virtually no sample preparation whatever. The second example, however, requires more involved treatment and when analyzing protein mixtures, the procedure can become very complex indeed involving extraction, centrifugation and fractional precipitation on reversed phases. In general, however, liquid samples become more difficult to prepare when the substances are present at very low concentrations. 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

Biological matrices are very complex apart from the analytes, they usually contain proteins, salts, aeids, bases, and various organie eompounds. Therefore, effeetive sample preparation must inelude partieulate eleanup to provide the component of interest in a solution, free from interfering matrix elements, and in an appropriate concentration. 

A general feature of optimum sample preparation is that maximum recovery of the analyte is observed. Consider a graph of recovery vs. variation in one experimental condition. Figure 5 shows such a graph, with temperature as the experimental variable. The curve exhibits a maximum and a decline on either side of the maximum. The assay will be most reproducible at the point of zero slope, i.e., at the maximum recovery, because small variations in conditions will not affect the result. In hydrolysis of a protein to its constituent amino acids, for example, it will be found that at very high temperatures or long hydrolysis times, degradation of the product amino acids occurs, while at low temperatures or short hydrolysis times, the protein... 

Dai, Y., Whittal R.M., and Li, L., Two-layer sample preparation a method for MALDI-MS analysis of complex peptide and protein mixtures, Anal. Chem., 71, 1087, 1999. 

Many process mixtures, notably fermentations, require sample preconcentration, microdialysis, microfiltration, or ultrafiltration prior to analysis. A capillary mixer has been used as a sample preparation and enrichment technique in microchromatography of polycyclic aromatic hydrocarbons in water.8 Microdialysis to remove protein has been coupled to reversed phase chromatography to follow the pharmacokinetics of the metabolism of acetaminophen into acetaminophen-4-O-sulfate and acetaminophen-4-O-glucu-ronide.9 On-line ultrafiltration was used in a process monitor for Aspergillus niger fermentation.10... 

One note of caution with regard to these types of studies is that one must take into account any other cellular mechanisms for substrate and/or product depletion. For example, in studies of enzymes that act on protein substrates, one must ensure that the substrate and products are isolated from cells under conditions that do not promote their destruction. In the case of kinases, for example, one must ensure that cellular phosphatases are inhibited during cell lysis and sample preparation, to ensure that substrate buildup is not the result of phosphatase-catalyzed dephosphorylation of the kinase product. 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Vaidyanathan, S. Winder, C. L. Wade, S. C. Kell, D. B. Goodacre, R. Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20kDa) proteins. Rapid Comm. Mass Spectrom. 2002,16,1276-1286. 

Encouraged by this spectral reproducibility, we focused our efforts on the particularly challenging problem of distinguishing bacterial strains by MALDI MS. We developed a modified correlation approach22 that relies on two fundamental qualities of bacterial mass spectra. First, because different bacterial strains of the same species have substantial, if not complete, genetic overlap, most of the protein masses observed with two different strains will be identical. This feature limits the value of the biomarker approach that is commonly used to differentiate bacteria species. Second, as just noted, closely controlled sample preparation and mass analysis procedures can result in highly reproducible results.22 The modified correlation approach takes advantage of subtle, yet reproducible, differences in mass spectra obtained from dif-... 

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