Method for the determination of conformation and conformational changes of proteins and of derivatives thereof

A method for the detection of a conformational state of a protein, said protein contained in a complex cell extract mixture of further proteins, other biomolecules, or both, comprising the following steps:

• limited proteolysis of a first sample of the complex cell extract mixture under a native condition, in which native condition the protein is in an original conformational state to be detected, leading to a first fragment sample;
• denaturation of the first fragment sample to a denaturated first fragment sample;
• enzymatic digestion of the denaturated first fragment sample to a first digested sample;
• analytical analysis of the first digested sample using at least one of selected reaction monitoring (SRM) and data-independent acquisition of product ion spectra;
• denaturation of a second sample of the complex cell extract mixture to a denaturated second fragment sample;
• enzymatic digestion of the denaturated second fragment sample to a second digested sample;
• analytical analysis of the second digested sample using at least one of selected reaction monitoring (SRM) and data-independent acquisition of product ion spectra;
• and
• determining the conformational state of the protein using a quantitative comparison of the analytical analysis of the first digested sample with the analytical analysis of the second digested sample.