Electrophoresis data interpretation

Comparing the mass spectra of the interaction experiments with those acquired from control experiments reveals differences that can be related to the structure of the interaction complex. Depending on the reaction, intact proteins, proteolytic peptides or both are simultaneously analyzed by MS and, optionally, MS/MS. If the sample is too complex for direct analysis, a broad range of additional means of separation are available, e.g., electrophoresis, LC, or affinity capture, which can all be efficiently combined with ESI or MALDI MS (Sects. 4.7 and 4.8). A major challenge for all three techniques described below is the data interpretation. This concerns less the identity of the resulting products than the molecular puzzle they create. Relating the observed differences between sample and control experiments to the structure of the interaction complex can be difficult, and great care is recommended before the data in hand are considered evidence for the existence of a certain structural element. 

FIG. 12.13 Net charge of egg albumin versus pH. The points were determined by electrophoresis, and the solid line by titration the broken line represents 60% of charge from titration. (Data from L. G. Longsworth, Ann. NY Acad. Sci., 41, 267 (1941). (Redrawn with permission from J. Th. G. Overbeek, Quantitative Interpretation of the Electrophoretic Velocity of Colloids. In Advances in Colloid Science, Vol. 3 (H. Mark and E. J. W. Verwey, Eds.), Wiley, New York 1950.)... 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

Figure 7.5 Analysis of the two three-way RNA junctions of the VS ribozyme by comparative gel electrophoresis. The secondary structure of the VS ribozyme is shown, with the sequences of the two component three-way junctions. Each was analyzed in isolation by comparative gel electrophoresis, comparing the mobilities of the three long-short arm species. As before, these species have a central core of RNA that is extended with DNA sections. The junction species were electrophoresed in 10% polyacrylamide gels in the presence of 90 mM Tris—borate (pH 8.3) with 3 (junction III—IV—V) or 5 (junction II—III—VI) mM Mg2. The structural interpretations of both sets of data are shown. Both junctions undergo coaxial stacking of two arms, with die third directed laterally.

A typical approach, in which the ionic strength is established by a 1-1 salt, and the pH is adjusted by an acid or base that has an ion in common with the inert electrolyte and is measured by means of a glass electrode, was used to study the following systems by titration, electrophoresis, and electroacoustics. A purely aqueous system was usually studied as a reference. The physical properties of solvents relevant to the interpretation of electrokinetic data (viscosity and permittivity) may be very different from those of water, and they have to be taken into account in the interpretation of results. 

While 2-D electrophoresis is fundamentally a very simple technique, numerous technical problems arise if the method is to give high resolution results which are reproducible and intercomparable, and if quantitative results are to be obtained. The interpretation of data posses additional challenges because the vast majority of proteins in both plant and animal tissues have been neither described nor named, and therefore have functions which remain to be discovered. 

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