pFind Studio: a computational solution for mass spectrometry-based proteomics
2022
Frontiers in Molecular Biosciences2022. Wu, Chun et al.
Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China
ABSTRACT:Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.
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Archives of Virology2022. Shi, YH et al.
Weifang Med Univ, Sch Life Sci & Technol, Weifang 261053, Peoples R China; Hunan Agr Univ, Hunan Prov Key Lab Biol & Control Plant Dis & Ins, Changsha 410128, Hunan, Peoples R China
ABSTRACT:Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups. In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.
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Biochemistry2022. S Shivakumaraswamy et al.
1Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064,India
ABSTRACT:
Use: pFind; pDeep; pLink
Nature2022. Ray, S et al.
Univ Sheffield, Hlth Lifespan & Neurosci Inst, Sheffield, S Yorkshire, England; Univ Bradford, Sch Pharm & Med Sci, Inst Canc Therapeut, Bradford, W Yorkshire, England; Univ Sheffield, Sch Biosci, Sheffield, S Yorkshire, England
ABSTRACT:Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation(1,2). Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.
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BIOCHEMISTRY2022. Shivakumaraswamy, S et al.
Jawaharlal Nehru Ctr Adv Sci Res, Mol Biol & Genet Unit, Bengaluru 560064, India
ABSTRACT:Guanosine 5 & PRIME;-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 & PRIME;-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being & LE;0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.
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Cell Metabolism2022. Runtsch, MC et al.
Trinity Coll Dublin, Sch Biochem & Immunol, Trinity Biomed Sci Inst, 152-160 Pearse St, Dublin D02 R590, Ireland
ABSTRACT:The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI. JAK1 activation was also inhibited by OI in response to IL-13, interferon-beta, and interferon-gamma in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation in vivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.
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Nature Communications2022. Zeng, Wen-Feng et al.
Univ Copenhagen, Fac Hlth Sci, NNF Ctr Prot Res, Prote Program, Copenhagen, Denmark; Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany
ABSTRACT:Machine learning and in particular deep learning (DL) are increasingly important in mass spectrometry (MS)-based proteomics. Recent DL models can predict the retention time, ion mobility and fragment intensities of a peptide just from the amino acid sequence with good accuracy. However, DL is a very rapidly developing field with new neural network architectures frequently appearing, which are challenging to incorporate for proteomics researchers. Here we introduce AlphaPeptDeep, a modular Python framework built on the PyTorch DL library that learns and predicts the properties of peptides (https://github.com/MannLabs/alphapeptdeep). It features a model shop that enables non-specialists to create models in just a few lines of code. AlphaPeptDeep represents post-translational modifications in a generic manner, even if only the chemical composition is known. Extensive use of transfer learning obviates the need for large data sets to refine models for particular experimental conditions. The AlphaPeptDeep models for predicting retention time, collisional cross sections and fragment intensities are at least on par with existing tools. Additional sequence-based properties can also be predicted by AlphaPeptDeep, as demonstrated with a HLA peptide prediction model to improve HLA peptide identification for data-independent acquisition (https://github.com/MannLabs/PeptDeep-HLA).
Use: pFind; pDeep
Cell2022. Yao, YK et al.
NIAID, Mol Dev Immune Syst Sect, Lab Immune Syst Biol & Clin Genom Program, NIH, Bethesda, MD 20892 USA; NCI, Expt Immunol Branch, NIH, Bethesda, MD 20892 USA
ABSTRACT:Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.
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Nature communications2022. Guan, L et al.
Univ Penn, Sch Arts & Sci, Dept Biol, Philadelphia, PA 19104 USA
ABSTRACT:The lack of tumor infiltration by CD8(+) T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8(+) T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8(+) T cells into tumors. In tissue samples from patients with melanoma, CD8(+) T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1(+) immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.Lack of CD8(+) T-cell infiltration into solid tumors is associated with poor responsiveness to immune checkpoint therapy (ICT). Here, the authors show that blocking the phosphorylation of HRS to reduce the induction of immunosuppressive exosomes promotes CD8(+) T-cell infiltration into tumors and enhances the efficacy of ICT in mouse melanoma models.
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Nature Communications2022. Fang, Z et al.
Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Dalian Univ Technol, Sch Bioengn, Dalian 116024, Peoples R China
ABSTRACT:Glycopeptides with unusual glycans or poor peptide backbone fragmentation in tandem mass spectrometry are unaccounted for in typical site-specific glycoproteomics analysis and thus remain unidentified. Here, we develop a glycoproteomics tool, Glyco-Decipher, to address these issues. Glyco-Decipher conducts glycan database-independent peptide matching and exploits the fragmentation pattern of shared peptide backbones in glycopeptides to improve the spectrum interpretation. We benchmark Glyco-Decipher on several large-scale datasets, demonstrating that it identifies more peptide-spectrum matches than Byonic, MSFragger-Glyco, StrucGP and pGlyco 3.0, with a 33.5%-178.5% increase in the number of identified glycopeptide spectra. The database-independent and unbiased profiling of attached glycans enables the discovery of 164 modified glycans in mouse tissues, including glycans with chemical or biological modifications. By enabling in-depth characterization of site-specific protein glycosylation, Glyco-Decipher is a promising tool for advancing glycoproteomics analysis in biological research.Poor peptide fragmentation and unusual glycan structures limit mass spectrometry-based analysis of intact N-glycopeptides. Here, the authors develop Glyco-Decipher, a glycan-independent peptide search tool, to tackle these issues and improve the coverage of site-specific glycan analysis.
Use: pFind; pGlyco; pQuant