pFind Studio: a computational solution for mass spectrometry-based proteomics
2024
Ecotoxicology and Environmental Safety2024. Cheng, Wanli et al.
National Key Laboratory of Agricultural Microbiology and National Engineering Research Center of Microbial Pesticides, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China
ABSTRACT:
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Cell Death Discovery2024. Dong, Xiaoshu et al.
State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, 518055, China
ABSTRACT:Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this study, we present a set of chemical probes that are derived from BITC, PEITC, and SFN and equipped with bioorthogonal alkynyl handles to systematically profile the target proteins of ITCs in live cancer cells. Using a competition-based quantitative chemical proteomics approach, we identify a range of candidate target proteins of ITCs enriched in biological processes such as apoptosis. We show that BID, an apoptosis regulator of the Bcl-2 family, is covalently modified by ITCs on its N-terminal cysteines. Functional characterization demonstrates that covalent binding to N-terminal cysteines of BID by PEITC results in conformational changes of the protein and disruption of the self-inhibitory interaction between N- and C-terminal regions of BID, thus unleashing the highly active C-terminal segment to exert downstream pro-apoptotic effects. Consistently, PEITC promotes the cleavage and mitochondrial translocation of BID, leading to a strong induction of apoptosis. We further show that mutation of N-terminal cysteines impairs the N- and C-terminal interaction of BID, relieving the self-inhibition and enhancing its apoptotic activity. Overall, our chemical proteomics profiling and functional studies not only reveal BID as the principal target of PEITC in mediating upstream events for the induction of apoptosis, but also uncover a novel molecular mechanism involving N-terminal cysteines within the first helix of BID in regulating its pro-apoptotic potential.
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Analytical Chemistry2024. Zhou, Keting et al.
MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 10084, China
ABSTRACT:Mapping disulfide linkages is crucial for characterizing pharmaceutical proteins during drug development and quality control. Traditional bottom-up protein analysis workflows often suffer from incomplete mapping for tryptic peptides consisting of multiple disulfide bonds. Although the employment of a partial reduction of disulfide bonds can improve disulfide mapping, it becomes a bottleneck of analysis because individual tuning is often needed. Herein, we have developed an online disulfide-ene reaction system in which the composition of the reaction solvent can be programmed to achieve optimal partial reduction of tryptic disulfide peptides after liquid chromatography separation. By coupling this system onto a bottom-up protein analysis workflow, high coverage for sequencing (71-83%) and disulfide mapping (84-100%) was achieved for standard proteins consisting of 4-19 disulfide bonds. The analytical capability was further demonstrated by mapping 13 scrambled disulfide bonds in lysozyme and achieving compositional analysis of IgG isotypes (kappa and lambda) and subclasses (IgG1-IgG4) from human plasma.
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Journal of Biological Chemistry2024. Zhao, Jianxin et al.
Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, China
ABSTRACT:O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus, and mitochondria. The recent advent of mass spectrometry technology has enabled the identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication, and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, etoposide, diminishes the association. Using higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon etoposide treatment and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress.
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Briefings in Bioinformatics2024. Ji, Mingjun et al.
Peking
ABSTRACT:Recent advances in neoantigen research have accelerated the development of immunotherapies for cancers, such as glioblastoma (GBM). Neoantigens resulting from genomic mutations and dysregulated alternative splicing have been studied in GBM. However, these studies have primarily focused on annotated alternatively-spliced transcripts, leaving non-annotated transcripts largely unexplored. Circular ribonucleic acids (circRNAs), abnormally regulated in tumors, are correlated with the presence of non-annotated linear transcripts with exon skipping events. But the extent to which these linear transcripts truly exist and their functions in cancer immunotherapies remain unknown. Here, we found the ubiquitous co-occurrence of circRNA biogenesis and alternative splicing across various tumor types, resulting in large amounts of long-range alternatively-spliced transcripts (LRs). By comparing tumor and healthy tissues, we identified tumor-specific LRs more abundant in GBM than in normal tissues and other tumor types. This may be attributable to the upregulation of the protein quaking in GBM, which is reported to promote circRNA biogenesis. In total, we identified 1057 specific and recurrent LRs in GBM. Through in silico translation prediction and MS-based immunopeptidome analysis, 16 major histocompatibility complex class I-associated peptides were identified as potential immunotherapy targets in GBM. This study revealed long-range alternatively-spliced transcripts specifically upregulated in GBM may serve as recurrent, immunogenic tumor-specific antigens.
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Journal of proteomics2024. Laku{\v{s}}i{\'c}, Margareta 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:Allopatric populations living under distinct ecological conditions are excellent systems to infer factors underlying intraspecific venom variation. The venom composition of two populations of Vipera ammodytes, , insular with a diet based on ectotherms and mainland with a diet based on ectotherms and endotherms, was compared considering the sex and age of individuals. Ten toxin families, dominated by PLA2, 2 , svMP, svSP, and DI, were identified through a bottom-up approach. The venom profiles of adult females and males were similar. Results from 58 individual SDS-PAGE profiles and venom pool analysis revealed significant differences between juveniles compared to subadults and adults. Two venom phenotypes were identified: a juvenile svMP-dominated and KUNlacking phenotype and an adult PLA2/svMP-balanced 2 /svMP-balanced and KUN-containing phenotype. Despite differences in prey availability (and, therefore, diet) between populations, no significant differences in venom composition were found. As the populations are geographically isolated, the lack of venom diversification could be explained by insufficient time for natural selection and/or genetic drift to act on the venom composition of island vipers. However, substantial differences in proteomes were observed when compared to venoms from geographically distant populations inhabiting different conditions. These findings highlight the need to consider ecological and evolutionary processes when studying venom variability. Significance: This study provides the first comprehensive analysis of the venom composition of two allopatric populations of Vipera ammodytes, , living under similar abiotic (climate) but distinct biotic (prey availability) conditions. The ontogenetic changes in venom composition, coupled with the lack of differences between sex and between populations, shed light on the main determinants of venom evolution in this medically important snake. Seven new proteomes may facilitate future comparative studies of snake venom evolution. This study highlights the importance of considering ecological and evolutionary factors to understand snake venom variation.
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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 Proteomics2024. 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 2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 2 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 mu g to 1000 mu g 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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Nature2024. Zhang, Zhenwei et al.
Cellular Biochemistry, Max-Planck-Institute for Multidisciplinary Sciences, Gttingen, Germany
ABSTRACT:Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5'splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal Bcomplex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-Bcomplex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of Bcomplexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5'splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
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Nature Communications2024. Michael, Andrew RM et al.
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N-4N1, Canada
ABSTRACT:
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