Peptide electrophoretic techniques

Despite the high efficiency of the present-day chromatographic and electrophoretic techniques and the fast developments in mass spectrometry, amino acid analysis still represents a valuable analytical tool in peptide chemistry for characterization of the final products, but also for monitoring intermediate coupling steps in solution and on solid supports by comparing ratios of diagnostic amino acids. 

Electrophoretic techniques, such as two-dimensional electrophoresis or isoelectric focusing, lend themselves under appropriate conditions to the separation with excellent resolution of small amounts of samples. They have mainly found use as powerful methods of analysis since their general applicability in the areas of peptide purification and isolation has, until recently, been severely restricted by limitations in sample capacity and instrumental design. 

Clark and Kricka have reviewed High-Resolution Analytical Techniques for Proteins and Peptides and Their Applications in Clinical Chemistry and include consideration of isotachophoresis, high-performance liquid chromatography, and high-resolution two-dimensional electrophoretic techniques for separation and analysis of complex protein mixtures. These techniques are not now widely used in clinical chemistry laboratories but represent the tools of the future, when laboratories will be required to measure gene products and the myriad proteins present, as in complex biologic fluids of significance in health and diseases. 

Capillary zone electrophoresis (CZE) is a powerful technique, magnetic in its analytical personality as a result of the simple way diverse analytes can be resolved rapidly and with high efficiency. The attraction is easy to understand—an electrophoretic technique with as much bandwidth as (and complementary to) HPLC and multiple modes of separation available by simply changing the buffer system. Yet within the simple instrumental framework that is, at its root, a power supply, a capillary and a detector, lies the capability to analyze drugs, peptides, carbohydrates, and proteins in sample matrices as simple as buffer or as complex as semm. That power is the magnet that draws people in. 

The molecular defect has recently been linked [7] to a single base substitution, an A—transversion, in the penultimate 3 nucleotide of the third intron of the Apo E gene. This leads to a loss of the correct 3 splice site, thus giving rise to two abnormally spliced mRNA forms. The smaUer form contains 53 nucleotides and the larger one, the entire third intron of the gene. Since both mRNA species contain chain termination codons within the intronic sequence, only short Apo E peptides not detectable by standard gel electrophoretic techniques are produced. Apo E deficiency is, therefore, the result of a molecular error which gives rise to shorter, nonfunctional forms of Apo E. In contrast to Apo B, where the mechanism is posttranslational, here it is clearly pretranslational. 

An example of the results obtained in the form of a chromatoelectropherogram can be seen in Figure 9.6. The contour type data display showed the three variables that were studied, namely chromatographic elution time, electrophoretic migration time, and relative absorbance intensity. Peptides were cleanly resolved by using this two-dimensional method. Neither method alone could have separated the analytes under the same conditions. The most notable feature of this early system was that (presumably) all of the sample components from the first dimension were analyzed by the second dimension, which made this a truly comprehensive multidimensional technique. 

In 1961 Ansorge et al. (A5), using the same technique as previously, determined the peptide composition of urine derived from four normal subjects, three males and one female. Among twenty isolated peptides, seventeen were found in all specimens of urine, two peptides in three specimens, and the remaining one only in two specimens of urine. The identity of individual peptides was established on the basis of their electrophoretic and chromatographic behavior, as well as the amino acid composition after complete hydrolysis. It should be pointed out, however, that the amino acid composition of the peptides examined differs considerably from that obtained by the same authors in the case of the peptides described in 1958 (HI). 

In theory, if the net charge, q, on a molecule is known, it should be possible to measure / and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define /by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. 

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

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