Probing protein unfolding through monitoring cysteine alkylation by matrix-assisted laser desorption/ionisation mass spectrometry.
Matrix-assisted laser desorption/ionisation mass spectrometry was used to monitor interaction between three proteins and two basic Immobiline chemicals (pK 10.3 and pK >12) commonly used in immobilised pH gradients (IPG). For two of the investigated proteins, the observed alkylation channels of the cysteine residues exhibited unmistakable response to their gradual denaturation following treatment with different concentrations (0-8 M) of two commonly used denaturants, urea and guanidine hydrochloride. Our assessment for protein unfolding is based on the number and relative intensity of the alkylation channels, yet the present mass spectrometry data are in good agreement with data based on optical rotatory dispersion, in which another approach was used to assess protein unfolding. Whether the present simple, fast and specific mass spectrometry method can be developed as a probe for monitoring folding/unfolding of cysteine-containing proteins can only be demonstrated by generating similar data for a larger number of proteins.
Galvani
M
M
GlaxoWellcome Medicines Research Center, via Fleming 4, Verona, Italy.
Hamdan
M
M
Righetti
P G
PG
D000478:Alkylation; D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry; D006736:Horses; D009113:Muramidase / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003574:Cytochrome c Group; D011506:Proteins; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D003545:Cysteine