Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylated proteins, analysis of selenomethionine incorporation, crystal content verification, and analysis of N-glycosylation site occupancy. Protocols for sample preparation, data collection, and analysis are given.
A recent development is the fluorescence-based thermal shift (ThermoFluor) assay. It uses an environmentally sensitive dye, Sypro Orange, to monitor the thermal stability of a protein and investigate factors (e.g., buffers, additives, and ligands) affecting this stability. This chapter describes the application of this method using a 96-condition in-house screen. The measurements are performed on a commercially available real-time PCR machine.
Multiangle or static light scattering (SLS) is a very powerful technique to determine the conformational state of proteins in solution, especially when used in combination with size exclusion chromatography (SEC). In the authors' experimental set-up the SLS detector is connected in-line to a standard protein purification machine (e.g., the Äkta Purifier) equipped with an analytical SEC column. The data collection and analysis are performed using commercial software.
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Acknowledgments
The work described here was supported by the MRC and the European Commission as SPINE, contract-no. QLG2-CT-2002-00988.
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Nettleship, J.E., Brown, J., Groves, M.R., Geerlof, A. (2008). Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light Scattering. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_19
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