Analysis of ubiquitinated proteome by quantitative mass spectrometry.
Protein modification by ubiquitin (Ub) is one of the most common posttranslational events in eukaryotic cells. Ubiquitinated proteins are destined to various fates such as proteasomal degradation, protein trafficking, DNA repair, and immune response. In the last decade, vast improvements of mass spectrometry make it feasible to analyze the minute amount of ubiquitinated components in vivo. When combined with quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC), it is capable of profiling ubiquitinated proteome under different experimental conditions. Here, we describe a procedure to perform such a study, including differential protein labeling by the SILAC method, enrichment of ubiquitinated species, mass spectrometric analysis, and quality control to reduce false positives. The potential challenges and limitations of the procedure are also discussed.
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Chan Hyun
CH
Na
Junmin
J
Peng
D000596:Amino Acids; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054875:Ubiquitination
D000596:Amino Acids; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D057149:Ubiquitinated Proteins; D014357:Trypsin