ABSTRACT: The aim of this experiment was to absolutely quantify adenylyl cyclase 8 (AC8) and the co-purified interactor calmodulin (CaM) to assess their stoichiometry.
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)
Project description:In data independent acquisition, consistent, comprehensive MS1 and MS2 can be recorded. This enables statistical comparison based on the two quantitative levels.
Project description:The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded on chromosome 5p. The patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infections. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but TNF-receptor NF-κB-signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts — but not leukocytes — facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies to α-toxin contribute to incomplete clinical penetrance. By disrupting cell-intrinsic immunity to α-toxin in non-leukocytic cells, human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease.
Project description:The standard proteomics database search strategy involves searching spectra against a peptide database and estimating the false discovery rate (FDR) of the resulting set of peptide-spectrum matches. One assumption of this protocol is that all the peptides in the database are reDR control strategies are needed. Recently, two methods were proposed to address this problem: subset-search and all-sub. We show that both methods fail to control the FDR. For subset-search, this failure is due to the presence of “neighbor” peptides, which are defined as irrelevant peptides with a similar precursor mass and fragmentation spectrum as a relevant peptide. Not considering neighbors compromises the FDR estimate because a spectrum generated by an irrelevant peptide can incorrectly match well to a relevant peptide. Therefore, we have developed a new method, “filter then subset-neighbor search” (FSNS), that accounts for neighbor peptides. We show evidence that FSNS properly controls the FDR when neighbors are present and that FSNS outperforms group-FDR, the only other method able to control the FDR relative to a subset of relevant peptides
Project description:In this study, label free peptide intensities from a well quantitatively characterised yeast cell lysate were acquired on two orbitrap mass spectrometers (LTQ-Velos and Q Exactive HF). Additionally, samples containing Universal Proteomics Standard and the Proteomics Dynamic range Standard (http://www.sigmaaldrich.com/life-science/proteomics/mass-spectrometry/ups1-and-ups2-proteomic.html) in a yeast background were also acquired. The absolute abundances of over 340 proteins present in that yeast lysate (determined in a parallel SRM-based study) were then used in order to determine the flyability (or detectability) of thousands of peptides. The flyability scores, termed the ‘F-factors’, reflect how well a peptide ionises in a complex chromatographically separated sample. Specifically, F-factors are calculated by normalising peptide precursor ion intensity by the absolute abundance of its parent protein. Based on the analysis of the six datasets deposited here (reflecting different gradients and instrument platforms) the study found that physicochemical properties including peptide length, charge and hydrophobicity are predictors of peptide detectability. Furthermore, it was established that hydrophobicity has a non-linear relationship with detectability and that coelution with competing ions significantly affects peptide detectability. The analysis based on the data deposited here suggests that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex chromatographically-separated peptide mixtures. The concept of F-factors will also undoubtedly assist in better surrogate selection in targeted mass spectrometry studies and allow more accurate calibration of peptide ion signal in label-free workflows.
Project description:The aim of the project was to characterize structural changes that occur on the rod cyclic nucleotide-gated channel upon interaction with calmodulin (CaM) directly in the native membrane.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The linear dynamic range and sensitivity of the microarray was measured by hybridizing dilution series of a Universal Reference (UR), an equimolar mixture of synthetic miRNAs. The UR was hybridized with 10,000 to 1 amol of each individual miRNA. Each individual miRNA and 1 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes was fluorescently labelled by 3â ligation. The RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool.
Project description:Picky is an online method designer for targeted proteomics. To assess the performance of PRM methods designed by Picky we carried out a benchmark experiment. As reference samples we used different amounts of human proteins spiked into 1.4 µg yeast lysate. These sample where analyzed in PRM and DDA mode. All methods inlcuded a shotgun method (top10 for DDA and top3 for PRM) and can therefore all be analyzed by MaxQuant.
Project description:Data independent acquisition (DIA) has become a well-established method in LC-MS driven proteomics. Nonetheless, there are still a lot of possibilities at the data analysis level. By benchmarking different DIA analysis workflows through a ground truth sample mimicking real differential abundance samples, consisting of a differential spike-in of UPS2 in a constant yeast background, we provide a roadmap for DIA data analysis of shotgun samples based on whether sensitivity, precision or accuracy is of the essence. Three different commonly used DIA software tools (DIA-NN, EncyclopeDIA and SpectronautTM) were tested in both spectral library mode and spectral library free mode. In spectral library mode we used the independent spectral library prediction tools Prosit and MS2PIP together with DeepLC, next to the classical DDA-based spectral libraries. In total we benchmarked 12 DIA workflows. DIA-NN in library free mode or using in silico predicted libraries shows the highest sensitivity maintaining a high reproducibility and accuracy.
Project description:Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, a second messenger involved in different signaling pathways. AC8 is one of the 9 membrane-bound isoforms, present in the brain and implicated in cognitive functions. AC8 is regulated by Ca2+/CaM and different G proteins but structural evidence on its regulation is scarce. We solved the structure of full-length AC8 in complex with Gas and CaM. ACs contain large stretches of disordered/highly flexible domains that cannot be resolved with cryo-EM. To overcome this limitation, we have studied AC8's interaction with CaM, Gas and Gbg using crosslinking mass spectrometry (XL-MS).
Project description:In this study, we generate genomic maps of Mediator, Rad2, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae. A related study involving ChIP-chip analysis of Rad2 occupany is also deposited at ArrayExpress under accession number E-MEXP-3875 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-3875 ).