Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering.
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 Akta Purifier) equipped with an analytical SEC column. The data collection and analysis are performed using commercial software.
Oxford Protein Production Facility, Oxford, UK.
Joanne E
JE
Nettleship
James
J
Brown
Matthew R
MR
Groves
Arie
A
Geerlof
D006031:Glycosylation; D008027:Light; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics
D011506:Proteins