Project description:In this study, we identified ubiquitinated peptides that changed abundance in the mutant of an E3 ubiquitin ligase gene by coupling a K-(GG) peptide immunoprecipitation with quantitative proteomic analysis. Two independent replicates identified 265 and 236 ubiquitinated peptides, respectively. Seventy-four ubiquitinated peptides were overlapped in both biological replicates. Quantitative analysis revealed that 27 of these ubiquitinated peptides changed abundance significantly (P < 0.05) in the mutant.
Project description:Using unbiased mass spectrometry-based screen to identify differences in ubiquitinated proteins in the overexpression of Usp11. To focus on potential substrates relevant for Usp11’s role in neurogenesis, we performed experiments with mouse embryonic stem (ES) cells, in which NPC and neurons are derived at Day8.
Project description:We developed a strategy that allows the identification of NEDP1 dependent NEDDylation sites under endogenous expression of wild type NEDD8. We combined the use of anti-diGly antibodies that recognise both ubiquitin and NEDD8 modified peptides upon trypsin digestion with short treatment of cells with the ubiquitin E1 inhibitor MLN7243 (UAEi), that dramatically reduces ubiquitin but not NEDD8 modification. By eliminating the majority of ubiquitin-derived diGly peptides upon MLN7243 treatment, we would be able to quantify NEDP1 dependent diGly peptides. Extracts from parental and NEDP1 knockout HCT116 cells both treated with UAEi were used for the isolation of diGly modified peptides and mass spectrometry analysis.
Project description:Protein function is regulated by post-translational modifications (PTMs) that may act individually or interact with others in a phenomenon termed PTM cross-talk. Multiple databases have been dedicated to PTMs, including recent initiatives oriented to the in silico prediction of PTM interactions. The study of PTM cross-talk ultimately requires experimental evidence about whether certain PTMs co-exist in a single protein molecule. However, available resources do not assist researchers in the experimental detection of co-modified peptides. Here we present TCellXTalk, a comprehensive database of phosphorylation, ubiquitination and acetylation sites in human T cells that supports the experimental detection of co-modified peptides using targeted or directed mass spectrometry. We demonstrate the efficacy of TCellXTalk and the strategy presented here in a proof of concept experiment that enabled the identification and quantification of 15 co-modified (phosphorylated and ubiquitinated) peptides in CD3 proteins of the T-cell receptor complex. To our knowledge, these are the first co-modified peptide sequences described in this widely studied cell type. Furthermore, quantitative data showed distinct dynamics of co-modified peptides upon T cell activation, demonstrating differential regulation of co-occurring PTMs in this biological context. Overall, TCellXTalk enables the experimental detection of co-modified peptides in human T cells and puts forward a novel and generic strategy for the study of PTM cross-talk.
Project description:To identify ubiquitinated modified proteins of lung adenocarcinoma, we collected five pairs of lung adenocarcinoma and normal lung tissues from the clinic for analysis
Project description:Arsenic contamination in food and ground water constitutes a public health concern to more than 100 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk in developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. In addition, we revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated with depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in ubiquitinated proteome in human cells, and found that the arsenite-elicited diminution of HMGCR ubiquitination involves TRC8.
Project description:This project was aimed to identify lipid droplet (LD) membrane proteins that are ubiquitylated. We isolated LDs from the livers of control and alcohol-fed rats and extracted LD membrane proteins.
Project description:Protein ubiquitination regulates key cellular functions including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show an ~3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.