Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry.
In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.
Haebel
S
S
Center for Biopolymers, University of Potsdam, Germany. haebel@rz.uni-potsdam.de
Albrecht
T
T
Sparbier
K
K
Walden
P
P
Körner
R
R
Steup
M
M
D000478:Alkylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D018698:Glutamic Acid / Q000737:chemistry*; D008745:Methylation; D006005:Phosphorylases / Q000737:chemistry; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis; D011198:Solanum tuberosum / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D016693:Receptors, Antigen, T-Cell, alpha-beta; D018698:Glutamic Acid; D006005:Phosphorylases