Improvement of ubiquitylation site detection by Orbitrap mass spectrometry

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-epsilon-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance: A K-epsilon-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. Indepth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular event

Distinct proteomic profiles in prefrontal subareas of elderly major depressive disorder and bipolar disorder patients

Abstract We investigated for the first time the proteomic profiles both in the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of major depressive disorder (MDD) and bipolar disorder (BD) patients. Cryostat sections of DLPFC and ACC of MDD and BD patients with their respective well-matched controls were used for study. Proteins were quantified by tandem mass tag and high-performance liquid chromatography-mass spectrometry system. Gene Ontology terms and functional cluster alteration were analyzed through bioinformatic analysis. Over 3000 proteins were accurately quantified, with more than 100 protein expressions identified as significantly changed in these two brain areas of MDD and BD patients as compared to their respective controls. These include OGDH, SDHA and COX5B in the DLPFC in MDD patients; PFN1, HSP90AA1 and PDCD6IP in the ACC of MDD patients; DBN1, DBNL and MYH9 in the DLPFC in BD patients. Impressively, depending on brain area and distinct diseases, the most notable change we found in the DLPFC of MDD was ‘suppressed energy metabolism’; in the ACC of MDD it was ‘suppressed tissue remodeling and suppressed immune response’; and in the DLPFC of BD it was differentiated ‘suppressed tissue remodeling and suppressed neuronal projection’. In summary, there are distinct proteomic changes in different brain areas of the same mood disorder, and in the same brain area between MDD and BD patients, which strengthens the distinct pathogeneses and thus treatment targets

Genome-wide DNA methylation profiling of non-small cell lung carcinomas

Background: Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions. Results: Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas. Conclusions: Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC

Measurements of nuclear level densities and gamma-ray strength functions and their interpretations

A method to extract primary $\gamma$-ray spectra from particle-$\gamma$ coincidences at excitation energies up to the neutron binding energy is described. From these spectra, the level density and $\gamma$-ray strength function can be determined. From the level density, several thermodynamical quantities are obtained within the microcanonical and canonical ensemble. Also models for the $\gamma$-ray strength function are discussed

Genome-wide DNA methylation profiling of non-small cell lung carcinomas

Abstract Background Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions. Results Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas. Conclusions Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.</p

The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation

One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex—in particular, the binding of the negative regulators Eto2 and Mtgr1—are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs