Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry.
The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.
Wang
Dongxia
D
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Xu
Wanping
W
McGrath
Sara C
SC
Patterson
Cam
C
Neckers
Len
L
Cotter
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D029721:Drosophila Proteins / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007477:Ions; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry*
C108740:Chi protein, Drosophila; D029721:Drosophila Proteins; D007477:Ions; D009687:Nuclear Proteins; D010455:Peptides; D025801:Ubiquitin; D014357:Trypsin; D019791:Guanidine; D005998:Glycine