The methanol-induced conformational transitions of beta-lactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry

J Am Soc Mass Spectrom. 2001 Mar;12(3):317-28. doi: 10.1016/s1044-0305(00)00226-9.


The methanol-induced conformational transitions under acidic conditions for beta-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with beta-sheets, alpha-helices, and both alpha-helices and beta-sheets, respectively, are studied under equilibrium conditions by electrospray ionization mass spectrometry (ESI-MS). The folding states of proteins in solution are monitored by the charge state distributions that they produce during ESI and by hydrogen/deuterium (H/D) exchange followed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induced alpha-helix structure. Intermediate states formed at about 35% methanol concentration are found to give bimodal charge state distributions. The same rate of H/D exchange is shown by the two contributions to the bimodal distributions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchange of proteins followed by ESI-MS shows that helical denatured states, populated at around 50% methanol concentration, transform into more protected structures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins.

MeSH terms

  • Cytochrome c Group / chemistry*
  • Disulfides / analysis
  • Hydrogen-Ion Concentration
  • Lactoglobulins / chemistry*
  • Methanol
  • Protein Conformation
  • Protein Denaturation
  • Solvents
  • Spectrometry, Mass, Electrospray Ionization
  • Ubiquitins / chemistry*


  • Cytochrome c Group
  • Disulfides
  • Lactoglobulins
  • Solvents
  • Ubiquitins
  • Methanol