El enfoque psicodramático de Elina Matoso y la perspectiva de la educación

Remarkable efficiencies of over a million plates per meter have been reported using capillary electrophoresis (CE) for protein separations [1-4] but only with pre-purified protein standards prepared in solvents. At least two issues must be resolved before CE can be applied to separate proteins in real biological samples. The first challenge is the wide dynamic range of protein expression in biological systems. Given the small sample injection volume of CE, a comprehensive, on-line enrichment step must be in place to allow for sufficient injection of the low abundance components. The second challenge is the complex nature of the biological sample background. In an electrophoretic based separation, the presence of abundant ionic salts is detrimental by causing band- broadening from excessive Joule heating and/or electrodispersion. The presence of salt increases the current flow which leads to increased heat, Joule heating, and changes the conductivity in a region of the capillary which changes the analyte mobility between regions causing peak tailing or fronting, electrodispersion.

An approach to address the above two issues is the use of solid phase mini-beds coupled on-line to CE [5]. In essence, a very short chromatography column packed with reversed phase beads is connected to the CE capillary for subsequent separation. The enrichment and salt removal is based on selective retention of proteins on reversed phase materials while the salt passes through unhindered. Using UV-visible absorption detection, the limit of detection (LOD) has been lowered 100-fold to the low ng mL-1 level for a mixture of standard peptides [6]. On-line mass spectral detection displayed reduced matrix effects with a LOD in the fmol mL-1 range for a complex mixture of peptides from a protein digest [7]. The major drawbacks of this method include the need for custom fabrication of the solid phase mini-bed, the challenge being to achieve high bed-to-bed reproducibility at such small dimensions, and the requirement for alterations to the CE instrument and/or custom designed components for the coupling.

Alternative enrichment techniques free of any solid phases have also been reported for CE and were discussed in Chapter 1, namely field-amplified sample stacking, pH-

mediated sample stacking, and isotachophoresis. Background matrix removal, however, must also be addressed.

Several attempts have been reported on overcoming problems associated with high ionic strength samples. Shihabi [8] performed sample stacking in the presence of salt by using acetonitrile to reduce the conductivity of the sample zone. Acetonitrile and other small organic solvents can act as terminating ions in transient pseudo isotachophoresis, allowing the sample to be separated in the presence of salt [9]. Other methods focused on the pH manipulation of the buffer solutions to enrich samples. These pH-mediated methods were less affected by the presence of salt in samples as compared to methods that rely solely on the conductivity of different regions. An example of a pH-mediated sample stacking method was reported by Park and Lunte [10] in which they created a zone of low conductivity between the sample plug and the acidic running buffer by electrokinetically injecting a plug of strong acid after the sample plug. Titration caused the catecholamine analytes to stack, regardless of the sample salt content [10]. A seven- fold increase in peak intensity was observed with no deterioration in separation efficiency in subsequent CZE (190,000 plates) [10]. This method, however, was only performed on small molecules with high mobilities, as opposed to larger peptides or proteins that have slower mobilities. A temporary pH junction between an acidic and basic solution, as opposed to a titration zone by a strong acid injection as above, was formed in the dynamic pH junction method developed by Britz-McKibbin et al. [11]. An acidified sample of nucleotides containing salt was and injected onto a capillary filled with a basic buffer, creating a pH junction between the sample zone and running buffer. A 50-fold enrichment was recorded for a mixture of nucleotides from a cell extract (including the naturally occurring salt levels). This dynamic pH junction method was then applied by Monton et al. [12] to the enrichment and separation of peptides. A 124-fold improvement in detection was observed. It is noteworthy that such pH junctions typically dissipate early in the run, and hence the above pH-mediated methods only allow for a small injection of a sample.

In contrast, we reported the use of a discontinuous buffer in CE to create a pH junction that endured through the entire run, permitting the entire capillary to be filled

with the sample solution. The capillary in this case was coated with a zwitterionic phospholipid bilayer [4] in order to prevent protein adsorption and to suppress the electroosmotic flow (EOF). Pre-purified protein standards were used to develop the discontinuous buffers method [13-15], but the effect of ionic salts must be explored to apply this method to real biological samples. It was speculated that ions possessing permanent charges, such as sodium and chloride, should maintain their mobilities when crossing the pH junction and thus eventually migrate out of the capillary.

In this chapter, the capability of discontinuous buffers in withstanding and removing non-buffering ionic salts present in the samples is investigated. Sodium chloride and myoglobin are used as the model salt and model analyte, respectively. In addition to in-capillary experiments with UV-absorbing ions, computer simulations were explored to provide further understanding to the ion mobility at the pH junction. Identification of the enriched and desalted protein sample is performed by offline matrix- assisted laser desorption ionization mass spectrometry (MALDI MS).

Following the methodology characterization based on a single protein (myoglobin), application on a protein mixture is also performed. This work builds on the existing discontinuous buffers protein enrichment and desalting operation by incorporating a subsequent capillary zone electrophoresis (CZE) separation. This coupling requires a reconfiguration of the buffer set-up following the enrichment and desalting step. A concentrated acid is introduced to remove the pH junction, and thus allow for the CZE separation.