NMR, X-ray crystallography, and calorimetry-based approaches typically require millimolar concentrations and milliliter volumes, hindering their use for proteins that are available only in low quantities and/or are difficult to purify. Furthermore, measuring affinity may require a concentration regime that is too low for determining the free energy of binding . Spectroscopy-based approaches such as fluorescence or circular dichroism generally require less sample, but when the binding is weak, these methods also require more sample . Owing to the high sensitivity of mass spectrometers and the chromatographic desalting and concentrating procedure in the protocol, PLIMSTEX is applicable to a wide range of protein concentrations by simply adjusting the amount of solution injected into the mass spectrometer. Small quantities (high picomole) and low concentration (nanomolar) of proteins are sufficient to obtain each point in a titration or kinetic run. Other direct or indirect MS methods (e.g. SUPREX ) also need only small amounts of protein.
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