Reversed phase protein extraction

The primary components of LCM technology are (1) visualization of the cells of interest through microscopy, (2) transfer of near-infrared laser energy pulses to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the polymer from the tissue surface, which shears the embedded cells of interest away from the heterogeneous tissue section (18,19). Extraction buffers applied to the polymer film solubilize the cells, liberating the molecules of interest. The DNA, RNA, or protein from the microdissected cells may be analyzed by any method with appropriate sensitivity (20,21,22,23,24). Protein extracted from microdissected cells may be used for mass spectrometric analysis, applied to reverse phase protein microarrays, or used for western blot analysis (25,26). 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

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 actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

Figure 9. The effect of storage on protein and peptide composition in cooked ground beef stored in a refrigerator of 4 days (adapted from 7). Upper graph represents the size exclusion chromatography of acidic extracts of fresh, cooked, and cooked-stored beef. Lx)wer graph represents the reverse phase HPLC of peak II from the size exclusion chromatography.

Despite their distinct advantages, on-line SPE and column-switching proce-dures do not always represent ideal separation techniques. In many cases, only a small number of samples can be analyzed before contamination of the precolumn by proteins occurs. Alternative techniques that prevent the adsorption of macromolecules onto column packings and allow direct injection of sample extracts are those based on use of specific LC columns. Shielded hydrophobic phase (27), small pore reversed-phase (28), and internal surface reversed-phase (29, 30) columns can be used to elute proteins in the excluded volumes, allowing small... 

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