Protein extraction studies, mass

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

High-throughput proteomic methods hold great promise for the discovery of novel protein biomarkers that can be translated into practical interventions for the diagnosis, treatment, and prevention of disease. These approaches may also facilitate the development of therapeutic agents that are targeted to specific molecular alterations in diseases such as cancer. In many cases, malignant cells yield unique protein profiles when total protein extracts from such cells are analyzed by 2-D gel electrophoresis or mass spectrometry (MS) methods. Such proteomic studies have the potential to provide an important complement to the analysis of DNA and mRNA extracts from these tissues.1... 

In principle, this increase in reproducibihty would allow for the detection of small features and/or differences between the mass spectra of closely related microorganisms. Assnming the direct transfer method has a variance similar to that of the dried-droplet method used in this study (Fig. 2.6), it can be inferred that a significant rednction in the variance can be realized by simply premixing the intact bacteria with the matrix solution prior to its deposition onto the MALDl plate, rather than performing sequential depositions of bacteria followed by matrix. This improvement in the reproducibility would also be expected to be observed in the protein extraction protocols, adding only a single dilution step in the overall procedure (1 1 sample/matrix). However, to date, all standard protocols (Freiwald and Sauer 2009) used in profile-based MALDI-MS analyses implement a dried-droplet approach to deposit the sample onto the MALDl plate. 

Centrifugal contacting devices have heen employed to demonstrate reverse micellar extraction of enzymes on an industrial scale (Dekker et al., 1991). Spray columns have heen employed in a number of studies to study mass transfer in such systems. The mass-transfer rate of protein lysozyme being extracted was satisfactorily described in terms of the mass-transfer coefficients of the reverse micelle (solvent) phase and the aqueous phase (Lye et al., 1996). Spray extraction columns have also been used to study the extraction of enzymes in aqueous two-phase systems (Arsalani et al., 2005). 

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