16 research outputs found
Differential protein expression profiling by ITRAQ-2DLC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor kiss-1 gene
Comunicaciones a congreso
Identificación de los mecanismos moleculares asociados con la toxicidad inducida por MeHg mediante el análisis de la expresión proteica diferencial
Comunicaciones a congreso
Stable isotopic labeling for proteomics
Since the coining of the term Proteome, the field of Proteomics has developed rapidly and come to rely heavily on mass spectrometry, initially for protein identification but recently the use of stable isotopic labels for protein quantitation has grown in importance. This trend has been driven by improvements in the mass spectrometers and reagents but also by the need to understand the molecular dynamics of cells. Since proteins are important effector molecules in most biochemical processes, key questions to be answered are: how much is present and when. In this chapter we describe the various methods currently available to quantitate proteins based on stable isotope protein labeling and discuss their merits as well as some of the issues still to be addressed
USP7 is a SUMO deubiquitinase essential for DNA replication
Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents
On the Statistical Significance of Compressed Ratios in Isobaric Labeling: A Cross-Platform Comparison
Isobaric
labeling is gaining popularity in proteomics due to its
multiplexing capacity. However, copeptide fragmentation introduces
a bias that undermines its accuracy. Several strategies have been
shown to partially and, in some cases, completely solve this issue.
However, it is still not clear how ratio compression affects the ability
to identify a protein’s change of abundance as statistically
significant. Here, by using the “two proteomes” approach
(<i>E. coli</i> lysates with fixed 2.5 ratios in the presence
or absence of human lysates acting as the background interference)
and manipulating isolation width values, we were able to model isobaric
data with different levels of accuracy and precision in three types
of mass spectrometers: LTQ Orbitrap Velos, Impact, and Q Exactive.
We determined the influence of these variables on the statistical
significance of the distorted ratios and compared them to the ratios
measured without impurities. Our results confirm previous findings− regarding the importance of optimizing acquisition parameters in
each instrument in order to minimize interference without compromising
precision and identification. We also show that, under these experimental
conditions, the inclusion of a second replicate increases statistical
sensitivity 2–3-fold and counterbalances to a large extent
the issue of ratio compression
Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation
Comprehensive
analysis of post-translational modifications (PTMs)
often depends on the purification of modified peptides prior to LC–MS/MS.
The implementation of these enrichment methods requires thorough knowledge
of the experimental conditions to achieve optimal selectivity and
sensitivity. In this regard, large-scale analysis of lysine acetylation,
a key PTM for multiple cellular processes, makes use of monoclonal
pan-antibodies designed against this moiety. We report that the immuno-purification
of lysine-acetylated peptides is hampered by the copurification of
lysine carbamylated peptides, a frequent urea artifact. This specific
interaction can be explained by the similar chemical structures of
lysine acetylation and lysine carbamylation. As an alternative, we
propose a sample preparation protocol based on sodium deoxycholate
that eliminates these artifacts and dramatically improves the selectivity
and sensitivity of this immuno-purification assay
A Proteomic Characterization of Factors Enriched at Nascent DNA Molecules
DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance