Whatever the application, MS-based analyses of FAC effluent will always be faced with the need to support a wide range of buffer components, ranging from variable ionic strength, surfactant levels to required cofactors. In select situations such as indicator analyses, online methods may be appropriate but it is clear that the insertion of an intermediate LC step offers significantly improved performance. This changes the nature of the data analysis, from the detection of sig-moidal breakthrough curves to peak detection and differential analysis across multiple fractions.
For online applications, breakthrough volume measurements are a simple matter of determining the inflection point when such curves are symmetrical, and a first derivative analysis can be useful in determining inflection points. Frequently the breakthrough curves are asymmetric at higher ligand concentration, where the binding isotherm can be nonlinear (see Fig. 6.4, for example). In this situation, the breakthrough volume is defined as the intensity-weighted midpoint. For offline applications involving LC/MS analysis of effluent fractions breakthrough curves are detected as peaks (Fig. 6.9) from extracted ion chromato-grams. Chromatographic peaks apparent in the control representing the unprocessed mixture but absent in an assay fraction indicates the presence of a hit; the quality of the hit is in turn determined by the fraction where compound breakthrough occurs. Automation of this process for large mixtures is necessary, as large volumes of data are generated. One approach involves the application of a component detection algorithm, to identify all features within an extracted ion chromatogram of significance, and then identifying differences between each feature and the control based on a user-defined tolerance (e.g. area differences > 10%). A requirement for successful ligand detection using this method is reproducibility in LC/MS, which we have shown is possible even with less-stable forms of chromatography such as HILIC . We note that software developed for quantitative proteomics applications perform similar functions, and can be co-opted for use in high-throughput FAC systems.
Typically, discovery-style high-throughput experiments are single-point determinations designed solely to identify hits rather than characterize the interactions. Infusions of isolated, newly discovered ligands over a concentration series provides the opportunity to map out the binding isotherm. Most frequently a single or multi-term Langmuir equation is sufficient to describe the data for straightforward binary interactions . Careful measurement of these isotherms, however, can reveal much about the nature of the binding event, and a simple Langmuir relationship need not be assumed. Affinity chromatography has been used to determine coupling constants between ligands exhibiting allosteric behavior, and FAC-MS represents an excellent method for making such measurements.
242 | 6 Frontal Affinity Chromatography - Mass Spectrometry for Ligand Discovery and Characterization 6.4
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