Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.
Advances in high resolution tandem mass spectrometry and peptide enrichment technologies have transformed the field of protein biochemistry by enabling analysis of end points that have traditionally been inaccessible to molecular and biochemical techniques. One field benefitting from this research has been the study of ubiquitin, a 76-amino acid protein that functions as a covalent modifier of other proteins. Seminal work performed decades ago revealed that trypsin digestion of a branched protein structure known as A24 yielded an enigmatic diglycine signature bound to a lysine residue in histone 2A. With the onset of mass spectrometry proteomics, identification of K-GG-modified peptides has emerged as an effective way to map the position of ubiquitin modifications on a protein of interest and to quantify the extent of substrate ubiquitination. The initial identification of K-GG peptides by mass spectrometry initiated a flurry of work aimed at enriching these post-translationally modified peptides for identification and quantification en masse. Recently, immunoaffinity reagents have been reported that are capable of capturing K-GG peptides from ubiquitin and its thousands of cellular substrates. Here we focus on the history of K-GG peptides, their identification by mass spectrometry, and the utility of immunoaffinity reagents for studying the mechanisms of cellular regulation by ubiquitin.
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080, USA.
Daisy
D
Bustos
Corey E
CE
Bakalarski
Yanling
Y
Yang
Junmin
J
Peng
Donald S
DS
Kirkpatrick
D006033:Glycylglycine / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D057149:Ubiquitinated Proteins; D054875:Ubiquitination
D010455:Peptides; D011506:Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D006033:Glycylglycine