pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
Communications Biology2023. Hesselbarth, Julia et al.
Martin Luther Univ Halle Wittenberg, Inst Biochem & Biotechnol, Interdisciplinary Res Ctr HALOmem, Charles Tanford Prot Ctr, Halle, Germany; Johannes Gutenberg Univ Mainz, Dept Chem Biochem, Bioctr 2, Mainz, Germany
ABSTRACT:The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1. Using native mass spectrometry, we identify the stoichiometry of sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce multimerisation of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role for membrane fusion. In summary, we unravel the stoichiometry of intermediates and off-pathway complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.Chemical cross-linking and native mass spectrometry aid the thorough investigation of complex formation and multimerisation of synaptic vesicle fusion-mediating SNARE complex, including the role of complexin-1 in its assembly.
Use: pLink
Cell Discovery2023. Yan, Junjun et al.
Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan, Hubei, Peoples R China; Huazhong Agr Univ, Natl Ctr Plant Gene Res, Hubei Hongshan Lab, Wuhan, Hubei, Peoples R China; Hubei Univ, Hubei Collaborat Innovat Ctr Green Transformat Bio, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn,Hubei Key, Wuhan, Hubei, Peoples R China
ABSTRACT:N-6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 angstrom) and S-adenosyl homocysteine (SAH)-bound (2.8 angstrom) states. Using an AI-empowered integrative approach based on AlphaFold prediction and chemical cross-linking mass spectrometry, we further modeled a near-complete structure of the quaternary complex. Coupled with biochemical characterization, we revealed that MTA1 serves as the catalytic core, MTA1, MTA9, and p1 likely accommodate the substrate DNA, and p2 may facilitate the stabilization of MTA1. These results together offer insights into the molecular mechanism underpinning methylation by the MTA1 complex and the potential diversification of MTases for N-6-adenine methylation.
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Proceedings of the National Academy of Sciences of the United States of America2023. Feng Yang et al.
School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
ABSTRACT:The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 A in diameter, connected via a three-section neck to an 840-A-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. Invitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.
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Journal of Proteome Research2023. Hoopmann, Michael R. et al.
Inst Syst Biol, Seattle, WA 98109 USA
ABSTRACT:Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.
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2023. Zhen, Xuhui et al.
Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:
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JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES2023. Bingkun Yang et al.
Hebei Med Univ, Inst Chinese Integrat Med, Sch Chinese Integrat Med, Dept Pharmacol Chinese Mat Med, 361,Zhongshan East Rd, Shijiazhuang 050017, Hebei, Peoples R China; Hebei Med Univ, Sch Pharm, Shijiazhuang 050017, Peoples R China
ABSTRACT:Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neuro-trophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent). Then the samples were analyzed using nanoLC-MS, followed by the identification of peptides using different sequence analysis software (PEAKS, pNovo and novor). Finally, bioinformatics analysis was performed to predict peptides with potential neuroprotective functions in CBL, such as anti-inflammatory and antioxidant peptides. Results showed that the number of peptides obtained by the MCX method coupled with PEAKS was the highest and the method was the most stable. Bioinformatic analysis of the detected peptides showed that two anti-inflammatory peptides (LLNLQPPPR and LSPSLRLP) and an antioxidant peptide (WPFPR) might be neuroprotective peptides in CBL. In addition, this study found that some peptides in CBL were present in myelin basic protein and tubulin beta chain. The results of this study for the detection of active peptides in CBL laid the foundation for the subsequent study of its active ingredients.
Use: pNovo
Briefings in Bioinformatics2023. Beslic, Denis et al.
BAM Fed Inst Mat Res & Testing, Richard Willstatter Str 11, D-12489 Berlin, Germany; Univ Potsdam, Digital Engn Fac, Hasso Plattner Inst, Prof Dr Helmert Str 2-3, D-14482 Potsdam, Germany; BAM Fed Inst Mat Res & Testing, Unter Eichen 87, D-12205 Berlin, Germany; Robert Koch Inst, ZKI PH 3,Nordufer 20, D-13353 Berlin, Germany
ABSTRACT:Monoclonal antibodies are biotechnologically produced proteins with various applications in research, therapeutics and diagnostics. Their ability to recognize and bind to specific molecule structures makes them essential research tools and therapeutic agents. Sequence information of antibodies is helpful for understanding antibody-antigen interactions and ensuring their affinity and specificity. De novo protein sequencing based on mass spectrometry is a valuable method to obtain the amino acid sequence of peptides and proteins without a priori knowledge. In this study, we evaluated six recently developed de novo peptide sequencing algorithms (Novor, pNovo 3, DeepNovo, SMSNet, PointNovo and Casanovo), which were not specifically designed for antibody data. We validated their ability to identify and assemble antibody sequences on three multi-enzymatic data sets. The deep learning-based tools Casanovo and PointNovo showed an increased peptide recall across different enzymes and data sets compared with spectrum-graph-based approaches. We evaluated different error types of de novo peptide sequencing tools and their performance for different numbers of missing cleavage sites, noisy spectra and peptides of various lengths. We achieved a sequence coverage of 97.69-99.53% on the light chains of three different antibody data sets using the de Bruijn assembler ALPS and the predictions from Casanovo. However, low sequence coverage and accuracy on the heavy chains demonstrate that complete de novo protein sequencing remains a challenging issue in proteomics that requires improved de novo error correction, alternative digestion strategies and hybrid approaches such as homology search to achieve high accuracy on long protein sequences.
Use: pNovo; pDeep
Journal of Proteome Research2023. Mengqi Luo et al.
Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041,
ABSTRACT:Human semen, consisting of spermatozoa (sperm) and seminal plasma, represents a special clinical sample type in human body fluid. Protein glycosylation in sperm and seminal plasma plays key roles in spermatogenesis, maturation, capacitation, sperm-egg recognition, motility of sperm, and fertilization. In this study, we profiled the most comprehensive O-glycoproteome map of human sperm and seminal plasma using our recently presented Glycoproteomics based on Two Complementary Fragmentation Methods (GlycoTCFM). We showed that sperm and seminal plasma contain many novel and distinctive O-glycoproteins, which are mostly located in the extracellular region (seminal plasma) and sperm membrane, enriched in the biological processes of cell adhesion and angiogenesis, and mainly involved in multiple biological functions including extracellular matrix structural constituents and binding. Based on GlycoTCFM, we created a comprehensive human sperm and seminal plasma O-glycoprotein database that contains 371 intact O-glycopeptides and 202 O-glycosites from 68 O-glycoproteins. Interestingly, 105 manually confirmed O-glycosites from 25 O-glycoproteins were reported for the first time, and they were mainly modified by core 1 O-glycans. We also found that three highly abundant, highly complex, and highly O-glycosylated proteins (semenogelin-1, semenogelin-2, and equatorin) may play important roles in sperm or seminal plasma composition and function. These data deepen our knowledge about O-glycosylation in sperm and seminal plasma and lay the foundation for the functional study of O-glycoproteins in male infertility.
Use: pGlyco
Proteomics2023. Chu-Wei Kuo et al.
Acad Sinica, Inst Biol Chem, 128 Acad Rd Sec 2, Taipei 115, Taiwan
ABSTRACT:Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.
Use: pGlyco
BIOLOGY-BASEL2023. Kaijun Chi et al.
Jiangnan Univ, Sch Biotechnol, Key Lab Carbohydrate Chem & Biotechnol, Minist Educ, Wuxi 214122, Peoples R China; Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
ABSTRACT:Simple Summary The Siglec-Fc protein, a fusion protein combining Siglec with the Fc part of a human antibody, is a promising sialic acid-Siglec axis-targeted agent for cancer treatment and is widely used for Siglec ligands discovery. The recombinant Siglec-Fc fusion protein has been expressed in different cell systems. However, its characteristics have not been investigated in detail. In this study, HEK293 and CHO cell lines were used to express the Siglec9-Fc protein, and their adaptability for production was compared. We optimized culture conditions and compared the glycosylation, yield, dimerization and sialic acid binding activity of the Siglec9-Fc protein produced in HEK293 and CHO. Using purified recombinant protein, we further analyzed the distribution of Siglec9 ligands on cancer cell lines, as well as bladder cancer tissue, and revealed the potential ligands. Our findings provide support for the selection of Siglec9-Fc protein expression systems and detection of related Siglec9 ligands. The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.
Use: pGlyco