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.