Diagram of GG-peptide enrichment strategy. (A) 8xHis-Ubiquitin is covalently attached to lysine residues of proteins. (B) Proteins are either mock treated or cleaved by addition of NTCB to the lysates. (C) Ubiquitinated proteins are purified by metal affinity resin. (D) Proteins are digested with trypsin. (E) Peptides are separated by strong cation exchange chromatography. (F) Peptides are identified by RPLC-MS/MS.

(A) Analysis of NTCB treatment and his-tag purifications of protein lysates from yeast strains either expressing 8xHis-ubiquitin or untagged ubiquitin by SDS-PAGE and coomassie staining. Lanes 1 and 2- whole cell lysates; lanes 3 and 4- protein fragments after treatment with NTCB; lanes 5 and 6- protein fragments post dialysis; lanes 7 and 8- metal affinity purified Ub-fragments. Yeast strains as indicated. (B) Validation of ubiquitinated protein kinases and phosphatases. The TAP strain for each of the indicated kinases and phosphatases was transformed with a 10xHis-ubiquitin expression plasmid and grown to mid-log phase. The yeast were lysed in denaturing buffer. 5% of the lysate was analyzed by anti-TAP western blot (left panel) and the 10xHis-ubiquitinated proteins were purified from the remaining lysate using metal affinity resin. The resin was boiled in sample buffer + ∀-mercaptoethanol and analyzed by anti-TAP western blot (middle panel; darker exposure in the right panel). Molecular weights are indicated. (C) The 13 yeast E2 ubiquitin conjugating enzymes aligned by ClustalW and colored according to percent identity, darker purple = higher conservation. The catalytic cysteine is highlighted with an arrow and the red boxes indicate the lysines that were identified as having a ubiquitin remnant mass shift in this study.