Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD.
View Article and Find Full Text PDFDysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes. Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSA) in comparison to wild type.
View Article and Find Full Text PDFThe transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs.
View Article and Find Full Text PDFSubstitution of protium (H) for deuterium (D) strongly affects biological systems. Whereas higher eukaryotes such as plants and mammals hardly survive a deuterium content of >30%, many microorganisms can grow on fully deuterated media, albeit at reduced rates. Very little is known about how the H/D replacement influences life at the systems level.
View Article and Find Full Text PDFMass spectrometry based proteomics has become the method of choice for pinpointing and monitoring thousands of post-translational modifications, predominately phosphorylation sites, in cellular signaling studies. Critical for achieving this analytical depth is the enrichment of phosphorylated peptides prior to liquid chromatography-mass spectrometry (MS) analysis. Despite the high prevalence of this modification, the numbers of identified phosphopeptides lag behind those achieved for unmodified peptides, and the cause for this still remains controversial.
View Article and Find Full Text PDFAlthough aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones.
View Article and Find Full Text PDFThe multiplexing capabilities of isobaric mass tag-based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of mass spectrometry-based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample prefractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion-based quantification has often been criticized for limited quantification accuracy due to interference from coeluting peptides and peptide fragments.
View Article and Find Full Text PDFMeasuring precise concentrations of proteins can provide insights into biological processes. Here we use efficient protein extraction and sample fractionation, as well as state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein-abundance map for Escherichia coli. We measure cellular protein concentrations for 55% of predicted E.
View Article and Find Full Text PDFThe data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5-10 times more sensitive than directed or DDA methods.
View Article and Find Full Text PDFWe evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells.
View Article and Find Full Text PDFComplex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies.
View Article and Find Full Text PDFThe generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale.
View Article and Find Full Text PDFConsiderable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function.
View Article and Find Full Text PDFSite-specific transcription factors (TFs) play an essential role in mammalian development and function as they are vital for the majority of cellular processes. Despite their biological importance, TF proteomic data is scarce in the literature, likely due to difficulties in detecting peptides as the abundance of TFs in cells tends to be low. In recent years, significant improvements in MS-based technologies in terms of sensitivity and specificity have increased the interest in developing quantitative methodologies specifically targeting relatively lowly abundant proteins such as TFs in mammalian models.
View Article and Find Full Text PDFIn recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC-MS/MS approaches. By focusing on specific peptide species, these methods allow hypothesis-driven analysis of selected proteins of interest, and they have been shown to be suited to monitor low-abundance proteins within complex mixtures. Despite their growing popularity, no study has systematically evaluated these various MS strategies in terms of quantification, detection, and identification limits when they are applied to complex samples.
View Article and Find Full Text PDFMolecular diversity of surface receptors has been hypothesized to provide a mechanism for selective synaptic connectivity. Neurexins are highly diversified receptors that drive the morphological and functional differentiation of synapses. Using a single cDNA sequencing approach, we detected 1,364 unique neurexin-α and 37 neurexin-β mRNAs produced by alternative splicing of neurexin pre-mRNAs.
View Article and Find Full Text PDFThe enteroinvasive bacterium Shigella flexneri invades the intestinal epithelium of humans. During infection, several injected effector proteins promote bacterial internalization, and interfere with multiple host cell responses. To obtain a systems-level overview of host signaling during infection, we analyzed the global dynamics of protein phosphorylation by liquid chromatography-tandem MS and identified several hundred of proteins undergoing a phosphorylation change during the first hours of infection.
View Article and Find Full Text PDFThere is a great interest in reliable ways to obtain absolute protein abundances at a proteome-wide scale. To this end, label-free LC-MS/MS quantification methods have been proposed where all identified proteins are assigned an estimated abundance. Several variants of this quantification approach have been presented, based on either the number of spectral counts per protein or MS1 peak intensities.
View Article and Find Full Text PDFWe report a high quality and system-wide proteome catalogue covering 71% (3,542 proteins) of the predicted genes of fission yeast, Schizosaccharomyces pombe, presenting the largest protein dataset to date for this important model organism. We obtained this high proteome and peptide (11.4 peptides/protein) coverage by a combination of extensive sample fractionation, high resolution Orbitrap mass spectrometry, and combined database searching using the iProphet software as part of the Trans-Proteomics Pipeline.
View Article and Find Full Text PDFThe complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis.
View Article and Find Full Text PDFThe relevance of libraries of annotated MS/MS spectra is growing with the amount of proteomic data generated in high-throughput experiments. These reference libraries provide a fast and accurate way to identify newly acquired MS/MS spectra. In the context of multiple hypotheses testing, the control of the number of false-positive identifications expected in the final result list by means of the calculation of the false discovery rate (FDR).
View Article and Find Full Text PDFMS2 library spectra are rich in reproducible information about peptide fragmentation patterns compared to theoretical spectra modeled by a sequence search tool. So far, spectrum library searches are mostly applied to detect peptides as they are present in the library. However, they also allow finding modified variants of the library peptides if the search is done with a large precursor mass window and an adapted Spectrum-Spectrum Match (SSM) scoring algorithm.
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