title: Using glycinylation, a chemical derivatization technique, for the quantitation of ubiquitinated proteins.
abstract: The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation. The most notable example is the use of deuteroacetylation to quantitate lysine acetylation. In this work, levels of lysine ubiquitination were quantitated using a structurally homologous label that is chemically similar to the diglycine (GlyGly) tag, which is left at the ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically equivalent monoglycine (Gly) tag that is analogous to the corresponding GlyGly tag proved that the monoglycine tag can be used for the quantitation of ubiquitination. A glycinylation protocol was then established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. Ubiquitin multimers were used to show that after glycinylation and tryptic digestion, the mass spectrometric response from the corresponding analogous tagged peptides could be compared for relative quantitation. For a proof of principle regarding the applicability of this technique to the analysis of ubiquitination in biological samples, the glycinylation technique was used to quantitate the increase in monoubiquitinated histone H2B that is observed in yeast which lacks the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast.
authors.affiliation: Johns Hopkins University School of Medicine, Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA. kstampe2@live.johnshopkins.edu
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Fiedler
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D057149:Ubiquitinated Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: D057149:Ubiquitinated Proteins; D005998:Glycine