The ubiquitin-proteasome pathway is the principal non-lysosomal pathway that controls the proteolysis of proteins. This pathway is significantly involved in a variety of cellular processes, including DNA repair, transcriptional regulation, signal transduction, cell metabolism and morphogenesis. The ubiquitination process is initiated when ubiquitin, an 8 kDa polypeptide consisting of 76 amino acids, is appended to cellular proteins via the C-terminal glycine of the ubiquitin molecule. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur. Polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin. Differences in total ubiquitination or the ubiquitination of specific proteins affect numerous pathological conditions, including malignancies, certain genetic diseases and neurodegenerative diseases.

1) Sample Preperation (Tissue lysis and Trypsinization):

Will require cell pellet or animal tissue. Approximatly 5-10 mg of proteins are extracted from the samples using the Guanidine based protein extraction method. Recovered proteins are denatured in 8M urea buffer and digested with trypsin. Peptides are then purified using solid phase extraction and quantified using BCA assay for Ubiquitome enrichment.

 2) Ubiquitome Enrichment using diGly Antibody:

We will use antibodies that recognize the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS).

3) LC-MS Analysis:

 The enriched peptides are run on a optimized 2 hour LC-MS run using a 25 cm C18 (BCH 1.7 micron) column using HCD fragmentation. Additional ETD fragmentation can be requested for additional charges. ETD fragmentation can increase the number of Ubiquitinated sites detected in the samples.