pFind Studio: a computational solution for mass spectrometry-based proteomics
2024
NATURE STRUCTURAL & MOLECULAR BIOLOGY2024. Sell{\'e}s-Baiget, Selene et al.
Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
ABSTRACT:Translesion DNA synthesis (TLS) is a cellular process that enables the bypass of DNA lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (Pol kappa) has the distinctive ability to bypass minor groove DNA adducts in vitro. However, Pol kappa is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base-editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Pol kappa during lesion bypass. Strikingly, we show that Pol kappa has two main functions during TLS, which are differentially regulated by Rev1 binding. On the one hand, Pol kappa is essential to replicate across a minor groove DNA lesion in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, through its cooperative interaction with Rev1 and ubiquitylated PCNA, Pol kappa appears to stabilize the Rev1-Pol zeta extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of Pol kappa's catalytic activity. Together, our work identifies catalytic and noncatalytic functions of Pol kappa in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases.The authors uncover the roles and regulations of DNA polymerase kappa (Pol kappa) during DNA damage bypass. In addition to a catalytic function across minor groove DNA lesions, Pol kappa stimulates Pol zeta-mediated extension past various DNA lesions.
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JOURNAL OF PROTEOME RESEARCH2024. Guo, Jisheng et al.
Research Department, The sixth peoples hospital of Zhengzhou, Zhengzhou 450000, China
ABSTRACT:Patients with cirrhosis face a heightened risk of complications, underscoring the importance of identification. We have developed a Connectome strategy that combines metabolites with peptide spectral matching (PSM) in proteomics to integrate metabolomics and proteomics, identifying specific metabolites bound to blood proteins in cirrhosis using open search proteomics methods. Analysis methods including Partial Least Squares Discriminant Analysis (PLS-DA), Uniform Manifold Approximation and Projection (UMAP), and hierarchical clustering were used to distinguish significant differences among the Cirrhosis group, Chronic Hepatitis B (CHB) group, and Healthy group. In this study, we identified 81 cirrhosis-associated connectomes and established an effective model distinctly distinguishing cirrhosis from chronic hepatitis B and healthy samples, confirmed by PLS-DA, hierarchical clustering analysis, and UMAP analysis, and further validated using six new cirrhosis samples. We established a Unified Indicator for Identifying cirrhosis, including tyrosine, Unnamed_189.2, thiazolidine, etc., which not only enables accurate identification of cirrhosis groups but was also further validated using six new cirrhosis samples and extensively supported by other cirrhosis research data (PXD035024). Our study reveals that characteristic cirrhosis connectomes can reliably distinguish cirrhosis from CHB and healthy groups. The established unified cirrhotic indicator facilitates the identification of cirrhosis cases in both this study and additional research data.
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NATURE GENETICS2024. Jang, H Josh et al.
Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA
ABSTRACT:Inhibiting epigenetic modulators can transcriptionally reactivate transposable elements (TEs). These TE transcripts often generate unique peptides that can serve as immunogenic antigens for immunotherapy. Here, we ask whether TEs activated by epigenetic therapy could appreciably increase the antigen repertoire in glioblastoma, an aggressive brain cancer with low mutation and neoantigen burden. We treated patient-derived primary glioblastoma stem cell lines, an astrocyte cell line and primary fibroblast cell lines with epigenetic drugs, and identified treatment-induced, TE-derived transcripts that are preferentially expressed in cancer cells. We verified that these transcripts could produce human leukocyte antigen class I-presented antigens using liquid chromatography with tandem mass spectrometry pulldown experiments. Importantly, many TEs were also transcribed, even in proliferating nontumor cell lines, after epigenetic therapy, which suggests that targeted strategies like CRISPR-mediated activation could minimize potential side effects of activating unwanted genomic regions. The results highlight both the need for caution and the promise of future translational efforts in harnessing treatment-induced TE-derived antigens for targeted immunotherapy.Treatment of primary glioblastoma cell lines with epigenetic therapy reactivates transposable elements (TEs). TE-derived transcripts can produce human leukocyte antigen class I-presented antigens, which could potentially be therapeutically targeted.
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CELL REPORTS2024. Liu, Zihua et al.
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
ABSTRACT:Itaconate serves as an immune-specific metabolite that regulates gene transcription and metabolism in both host and pathogens. S-itaconation is a post-translational modification that regulates immune response; however, its antimicrobial mechanism under the physiological condition remains unclear. Here, we apply a bioorthogonal itaconate probe to perform global profiling of S-itaconation in living pathogens, including S. Typhimurium, S. aureus, , and P. aeruginosa. . Some functional enzymes are covalently modified by itaconate, including those involved in the de novo purine biosynthesis pathway. Further biochemical studies demonstrate that itaconate suppresses this specific pathway to limit Salmonella growth by inhibiting the initiator purF to lower de novo purine biosynthesis and simultaneously targeting the guaABC cluster to block the salvage route. Our chemoproteomic study provides a global portrait of S-itaconation in multiple pathogens and offers a valuable resource for finding susceptible targets to combat drug-resistant pathogens in the future.
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Experimental Technology & Management2024. et al.
ABSTRACT:
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NATURE COMMUNICATIONS2024. Jiang, Yida et al.
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
ABSTRACT:While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism. Our results reveal a two-step pathway with mainly sequential generation of diazo and carbene intermediates. Diazo intermediate preferentially targets buried polar residues, many of which are inaccessible with known CXL probes for their limited reactivity. Moreover, we demonstrate that tuning light intensity and duration enhances selectivity towards polar residues by biasing diazo-mediated cross-linking reactions over carbene ones. This mechanistic dissection unlocks the full potential of PXL, paving the way for accurate distance mapping against protein structures and ultimately, unveiling protein dynamic behaviors.Photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, but unambiguous identification of cross-linked residues hinders data interpretation. Here, the authors report a quantitative analysis of alkyl diazirine photo-cross-linking reactions and reveal a two-step mechanism, enabling selective targeting of buried polar residues.
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Biochimie2024. Damm, Maik et al.
CIBIO, Centro de Investigao em Biodiversidade e Recursos Genticos, InBIO Laboratrio Associado, Campus de Vairo, Universidade do Porto, 4485-661, Vairo, Portugal
ABSTRACT:The North African mountain viper (Vipera monticola) is a medically relevant venomous snake distributed in Morocco, Algeria, and Tunisia. Three subspecies of V.monticola, exhibiting differences in morphotypes and dietary regimes, are currently recognised: V.m. monticola, V.m. atlantica, and V.m. saintgironsi. Through the application of snake venomics, we analysed the venoms of specimens of Moroccan origin belonging to each of the three subspecies. Snake venom metalloproteinases (svMP), snake venom serine proteases (svSP), C-type lectin and C-type lectin-related proteins (CTL), and phospholipases A2 (PLA2) were predominant, with PLA2 being the most abundant toxin family overall. Disintegrins (DI) and cysteine-rich secretory proteins (CRISP) were exclusive to V.m. monticola and V.m. atlantica, while l-amino-acid oxidases (LAAO) were only found in V.m. saintgironsi. The differences detected in the venom profiles, as well as in presence/absence and relative abundances of toxin families, indicate the occurrence of intraspecific venom variation within V.monticola. The identified patterns of venom similarity between subspecies seem to align more with their phylogenetic relationships than with the reported differences in their feeding habits.
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Molecular & cellular proteomics : MCP2024. Shao, Xianfeng et al.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
ABSTRACT:Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 mug to 1000 mug of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples.
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JOURNAL OF PROTEOME RESEARCH2024. Damm, Maik et al.
Institut fr Chemie, Technische Universitt Berlin, Strae des 17. Juni 135, 10623 Berlin, Germany
ABSTRACT:Snake venom variations are a crucial factor to understand the consequences of snakebite envenoming worldwide, and therefore it is important to know about toxin composition alterations between taxa. Palearctic vipers of the genera Vipera, Montivipera, Macrovipera, and Daboia have high medical impacts across the Old World. One hotspot for their occurrence and diversity is Turkiye, located on the border between continents, but many of their venoms remain still understudied. Here, we present the venom compositions of seven Turkish viper taxa. By complementary mass spectrometry-based bottom-up and top-down workflows, the venom profiles were investigated on proteomics and peptidomics level. This study includes the first venom descriptions of Vipera berus barani, Vipera darevskii, Montivipera bulgardaghica albizona, and Montivipera xanthina, as well as the first snake venomics profiles of Turkish Macrovipera lebetinus obtusa, and Daboia palaestinae, including an in-depth reanalysis of M. bulgardaghica bulgardaghica venom. Additionally, we identified the modular consensus sequence pEXW(PZ)1-2P(EI)/(KV)PPLE for bradykinin-potentiating peptides in viper venoms. For better insights into variations and potential impacts of medical significance, the venoms were compared against other Palearctic viper proteomes, including the first genus-wide Montivipera venom comparison. This will help the risk assessment of snakebite envenoming by these vipers and aid in predicting the venoms' pathophysiology and clinical treatments.
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Journal of Proteome Research2024. Wei, Qianzhou et al.
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and MOE Key Laboratory of Tumor Molecular Biology, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China
ABSTRACT:The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify all proteins encoded by the human genome. Currently, the human proteome still contains approximately 2000 PE2-PE5 proteins, referring to annotated coding genes that lack sufficient protein-level evidence. During the past 10 years, it has been increasingly difficult to identify PE2-PE5 proteins in C-HPP approaches due to the limited occurrence. Therefore, we proposed that reanalyzing massive MS data sets in repository with newly developed algorithms may increase the occurrence of the peptides of these proteins. In this study, we downloaded 1000 MS data sets via the ProteomeXchange database. Using pFind software, we identified peptides referring to 1788 PE2-PE5 proteins. Among them, 11 PE2 and 16 PE5 proteins were identified with at least 2 peptides, and 12 of them were identified using 2 peptides in a single data set, following the criteria of the HPP guidelines. We found translation evidence for 16 of the 11 PE2 and 16 PE5 proteins in our RNC-seq data, supporting their existence. The properties of the PE2 and PE5 proteins were similar to those of the PE1 proteins. Our approach demonstrated that mining PE2 and PE5 proteins in massive data repository is still worthy, and multidata set peptide identifications may support the presence of PE2 and PE5 proteins or at least prompt additional studies for validation. Extremely high throughput could be a solution to finding more PE2 and PE5 proteins.
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