Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.
Peng
Jianhe
J
Institut für Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, Germany. jpeng@gwdg.de
Schmidt
Bernhard
B
von Figura
Kurt
K
Dierks
Thomas
T
D000409:Alanine / Q000031:analogs & derivatives* / Q000032:analysis; D000595:Amino Acid Sequence; D005998:Glycine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D007709:Klebsiella / Q000201:enzymology; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013429:Sulfatases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
C524739:C(alpha)-formylglycine; D013429:Sulfatases; D014357:Trypsin; D000409:Alanine; D005998:Glycine