Molecular & Cellular Proteomics最新文献

Metaproteomics, the study of collective proteomes in environmental communities, plays a crucial role in understanding microbial functionalities affecting ecosystems and human health. Pathway analysis offers structured insights into the biochemical processes within these communities. However, no existing tool effectively combines pathway analysis with peptide- or protein-level data. We here introduce PathwayPilot, a web-based application designed to improve metaproteomic data analysis by integrating pathway analysis with peptide- and protein-level data, filling a critical gap in current metaproteomics bioinformatics tools. By allowing users to compare functional annotations across different samples or multiple organisms within a sample, PathwayPilot provides valuable insights into microbial functions. In the re-analysis of a case study examining the effects of caloric restriction on gut microbiota, the tool successfully identified shifts in enzyme expressions linked to short-chain fatty acid biosynthesis, aligning with its original findings. PathwayPilot's user-friendly interface and robust capabilities make it a significant advancement in metaproteomics, with potential for widespread application in microbial ecology and health sciences. All code is open source under the Apache2 license and is available at https://pathwaypilot.ugent.be.

Genotype-phenotype correlations of rare diseases are complicated by low patient number, high phenotype variability and compound heterozygosity. Mutations may cause instability of single proteins, and affect protein complex formation or overall robustness of a specific process in a given cell. Ciliopathies offer an interesting case for studying genotype-phenotype correlations as they have a spectrum of severity and include diverse phenotypes depending on different mutations in the same protein. For instance, mutations in the intraflagellar transport protein IFT140 cause a vast spectrum of ciliopathies ranging from isolated retinal dystrophy to severe skeletal abnormalities and multi-organ diseases such as Mainzer-Saldino and Jeune syndrome. Here, the quantitative effects of 23 missense mutations in IFT140, which forms part of the crucial IFT-A complex of the ciliary machinery, were analyzed using affinity purification coupled to mass spectrometry (AP-MS). A subset of 10 mutations led to a significant and domain-specific reduction in IFT140-IFT-A complex interaction indicating complex formation issues and potentially hampering its molecular function. Knockout of IFT140 led to loss of cilia, as shown before. However, phenotypically only mild effects concerning cilia assembly were observed for two out of four tested IFT140 missense mutations. Therefore, our results demonstrate the utility of AP-MS in discerning pathogenic MMs from polymorphisms, and we postulate that reduced function is tolerated by the evolutionarily highly conserved IFT-A system.

Mass spectrometry-based proteomics has revolutionized bacterial identification and elucidated many molecular mechanisms underlying bacterial growth, community formation, and drug resistance. However, most research has been focused on a few model bacteria, overlooking bacterial diversity. In this study, we present the most extensive bacterial proteomic resource to date, covering 303 species, 119 genera, and five phyla with over 636,000 unique expressed proteins, confirming the existence of over 38,700 hypothetical proteins. Accessible via the public resource ProteomicsDB, this dataset enables quantitative exploration of proteins within and across species. Additionally, we developed MS2Bac, a bacterial identification algorithm that queries NCBI's bacterial proteome space in two iterations. MS2Bac achieved over 99% species-level and 89% strain-level accuracy, surpassing methods like MALDI-TOF and FTIR, as demonstrated with food-derived bacterial isolates. MS2Bac also effectively identified bacteria in clinical samples, highlighting the potential of MS-based proteomics as a routine diagnostic tool.

Accurate genome duplication requires a tightly regulated DNA replication program, which relies on the fine regulation of origin firing. While the molecular steps involved in origin firing have been determined predominantly in budding yeast, the complexity of this process in human cells has yet to be fully elucidated. Here, we describe a straightforward proteomics approach to systematically analyse protein recruitment to the chromatin during induced origin firing in human cells. Using a specific inhibitor against CHK1 kinase, we induced a synchronised wave of dormant origin firing (DOF) and assessed the S phase chromatin proteome at different time points. We provide time-resolved loading dynamics of 3,269 proteins, including the core replication machinery and origin firing factors. This dataset accurately represents known temporal dynamics of proteins on the chromatin during the activation of replication forks and the subsequent DNA damage due to the hyperactivation of excessive replication forks. Finally, we used our dataset to identify the condensin II subunit NCAPH2 as a novel factor required for efficient origin firing and replication. Overall, we provide a comprehensive resource to interrogate the protein recruitment dynamics of replication origin firing events in human cells.

Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote concerted response mechanisms remain understudied. Here, we show that K63-linked polyubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 2.5-fold the pool of proteins (2,494) and provided a comprehensive number of sites (10,157) known to be ubiquitinated during arsenite stress, suggesting their involvement in a myriad of cellular pathways. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.

Intercellular communication is fundamental to multicellular life and a core determinant of outcomes during viral infection, where the common goals of virus and host for persistence and replication are generally at odds. Hosts rely on encoded innate and adaptive immune responses to detect and clear viral pathogens, while viruses can exploit or disrupt these pathways and other intercellular communication processes to enhance their spread and promote pathogenesis. While virus-induced signaling can result in systemic changes to the host, striking alterations are observed within the cellular microenvironment directly surrounding a site of infection, termed the virus microenvironment (VME). Mechanisms employed by viruses to condition their VMEs are emerging and are critical for understanding the biology and pathologies of viral infections. Recent advances in experimental approaches, including proteomic methods, have enabled study of the VME in unprecedented detail. In this review article, we provide a primer on proteomic approaches used to study how viral infections alter intercellular communication, highlighting the ways in which these approaches have been implemented and the exciting biology they have uncovered. First, we consider the different molecules secreted by an infected cell, including proteins, either soluble or contained within extracellular vesicles, and metabolites. We further discuss the modalities of interactions facilitated by alteration at the cell surface of infected cells, including immunopeptide presentation and interactions with the extracellular matrix. Finally, we review spatial profiling approaches that have allowed distinguishing how specific subpopulations of cells within a VME respond to infection and alter their protein composition, discussing valuable insights these methods have offered.

Single cell proteomics was performed on human induced pluripotent stem cells (iPSCs), iPSC-derived cardiomyocytes, and adult cardiomyocytes. Over 700 proteins could be simultaneously measured in each cell revealing unique subpopulations. A sub-set of iPSCs expressed higher levels of Lin28a and Tra-1-60 towards the outer edge of cell colonies. In the cardiomyocytes, two distinct populations were found that exhibited complementary metabolic profiles. Cardiomyocytes from iPSCs showed a glycolysis profile while adult cardiomyocytes were enriched in proteins involved with fatty acid metabolism. Interestingly, rare single cells also co-expressed markers of both cardiac and neuronal lineages, suggesting there maybe a novel hybrid cell type in the human heart.

Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation.

Signaling pathways often convergence on transcription factors (TFs) and other DNA-binding proteins (DBPs) that regulate chromatin structure and gene expression, thereby governing a broad range of essential cellular functions. However, the repertoire of DBPs is incompletely understood even for the best-characterized pathways. Here, we optimized a strategy for the isolation of Proteins on Chromatin (iPOC) exploiting tagged nucleoside analogues to label the DNA and capture associated proteins, thus enabling the comprehensive, sensitive, and unbiased characterization of the DNA-bound proteome. We then applied iPOC to investigate chromatome changes upon perturbation of the cancer-relevant PI3K/AKT/mTOR pathway. Our results show distinct dynamics of the DNA-bound proteome upon selective inhibition of PI3K, AKT, or mTOR, and we provide evidence how this signaling cascade regulates the DNA-bound status of SUZ12, thereby modulating H3K27me3 levels. Collectively, iPOC is a powerful approach to study the composition of the DNA-bound proteome operating downstream of signaling cascades, thereby both expanding our knowledge of the mechanism of action of the pathway, and unveiling novel chromatin modulators that can potentially be targeted pharmacologically.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC₅₀ of 17 nM and EphB4 with an IC₅₀ of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC₅₀ of 2.1 nM, suggesting that a DDR1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on non-kinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.