Abstract
Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.
Highlights
The structural investigation of protein assemblies has become important for the understanding of cellular processes
Mass spectrometry considerably contributes to the structural investigation in that it can be applied to almost every complex of interest irrespective of size or sample heterogeneity
After in-gel digestion of the proteins, the peptide mixture was analyzed by liquid chromatography-coupled mass spectrometry and peptide and fragment masses were subjected to database searching
Summary
The structural investigation of protein assemblies has become important for the understanding of cellular processes. In combination with ion mobility, native mass spectrometry further allows determination of their conformation[23,24] This makes it a powerful tool for the structural investigation of protein complexes that are difficult to assess by conventional. Native mass spectrometry requires analysis buffers which maintain non-covalent protein interactions during electrospray ionization This is usually achieved by using aqueous, volatile buffers such as ammonium acetate[25]. The stoichiometries of chaperone complexes, including an unexpected Hsp[70] dimer, could be obtained from mass spectrometry experiments of the intact protein complexes, while chemical cross-linking revealed the arrangements of the proteins in the assemblies[30,31]. Protein complexes purified for structural analysis usually have the quality required for successful analysis with our protocols
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