Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins.
Post-translational modifications of proteins including phosphorylation, glycosylation, acetylation and ubiquitination facilitate the regulation of many cellular processes and intracellular signaling events. Ubiquitination plays a key role in the functional regulation and degradation of many classes of proteins, and the study of ubiquitination and poly-ubiquitination has emerged as one of the most active areas in proteomic research. A variety of mass spectrometric methods have been described for the identification of ubiquitination sites, the study of poly-ubiquitin topology and the identification of ubiquitin substrates. The most popular workflow for both ubiquitination site mapping and poly-ubiquitination chain topology characterization is to take advantage of the Gly-Gly signature on the substrate's lysine residue observed after tryptic digestion. Although a number of protocols have been described for the mapping of ubiquitination sites, one major challenge is that ubiquitination is typically heterogeneous, and several lysine residues may be ubiquitinated within a protein. When multiple ubiquitination sites are present, multiple analyses are often required to cover all of the potential modification sites which in turn can necessitate the usage of larger quantities of material. In addition, the level of ubiquitination on endogenous and recombinant proteins may be of low intensity, adding further analytical challenges in the identification of this modification. The use of the multiple reaction monitoring (MRM)-initiated detection and sequencing workflow (MIDAS) for the identification of phosphorylation sites has previously been described. Here, we explore the use of an MRM workflow for ubiquitination site mapping on the substrate protein, receptor interacting protein (RIP).
Mollah
Sahana
S
Applied Biosystems, Foster City, CA 94404, USA.
Wertz
Ingrid E
IE
Phung
Qui
Q
Arnott
David
D
Dixit
Vishva M
VM
Lill
Jennie R
JR
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D025801:Ubiquitin; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2