pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
Biochemistry2023. liuhaijun et al.
Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States
ABSTRACT:Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod-core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment-protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane. Here, the structural synergy between the rod-core region and the core-membrane region was investigated using quantitative mass spectrometry (MS). The AlphaFold-predicted CpcG-CTE structure was first modeled onto the PBS rod-core region, guided and justified by the isotopically encoded structural MS data. Quantitative cross-linking MS analysis revealed that the structural proximity of the PB-loop in ApcE and ApcG-CTE is significantly disturbed in the absence of six PBS rods, which are attached to PBS via CpcG-CTE, indicative of drastic conformational changes and decreased structural integrity. These results suggest that CpcG-rod attachment on the PBS rod-core side is essentially required for the PBS core-membrane structural assembly. The hypothesized long-range synergy between the rod-core interface (where the orange carotenoid protein also functions) and the terminal energy emitter of PBS must have important regulatory roles in PBS core assembly, light-harvesting, and excitation energy transmission. These data also lend strategies that genetic truncation of the light-harvesting antennas aimed for improved photosynthetic productivity must rely on an in-depth understanding of their global structural integrity.
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Journal of Proteome Research2023. Sun, Zhen et al.
Anhui Med Univ, Hefei 230032, Peoples R China; Chinese Acad Med Sci, Natl Ctr Prot Sci Beijing, Res Unit Prote Res & Dev New Drug, State Key Lab Prote,Beijing Proteome Res Ctr,Inst, Beijing 102206, Peoples R China; Wuhan Univ, Sch Basic Med Sci, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery,Mi, Wuhan 430071, Peoples R China; Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Divers, Baoding 071002, Hebei, Peoples R China
ABSTRACT:Trypsin specifically cleaves the C-terminus of lysine and arginine residues but often fails to cleave modified lysines, such as ubiquitination, therefore resulting in the uncleaved K-e-GG peptides. Therefore, the cleaved ubiquitinated peptide identification was often regarded as false positives and discarded. Interestingly, unexpected cleavage at the K48-linked ubiquitin chain has been reported, suggesting the latent ability of trypsin to cleave ubiquitinated lysine residues. However, it remains unclear whether other trypsin-cleavable ubiquitinated sites are present. In this study, we verified the ability of trypsin in cleaving K6 and K63 besides K48 chains. The uncleaved K-e-GG peptide was quickly and efficiently generated during trypsin digestion, whereas cleaved ones were produced with much lower efficiency. Then, the K-e- GG antibody was proved to efficiently enrich the cleaved K-e-GG peptides and several published large-scale ubiquitylation datasets were re-analyzed to interrogate the cleaved sequence features. In total, more than 2400 cleaved ubiquitinated peptides were identified in the K-e-GG and UbiSite antibody-based datasets. The frequency of lysine upstream of the cleaved modified K was significantly enriched. The kinetic activity of trypsin in cleaving ubiquitinated peptides was further elucidated. We suggest that the cleaved K-e-GG sites with high post-translational modification probability (>= 0.75) should be considered as true positives in future ubiquitome analyses.
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Cell2023. Qin, Fangfei et al.
Shenzhen Bay Lab, Shenzhen 518055, Peoples R China; Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, Sch Life Sci, Key Lab Cell Proliferat & Differentiat, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Beijing 100871, Peoples R China
ABSTRACT:A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and b-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr0s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a plat-form technology for elucidating the "metabolites-modification-regulation"axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
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The Journal of biological chemistry2023. Deng, Hongyan et al.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, P. R. China
ABSTRACT:The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide-induced cell death and Cystine starvation/Erastin-induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity.
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Journal of Proteome Research2023. Gao, Jing et al.
Analytical Research Center for Organic and Biological Molecules, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Media, Chinese Academy of Sciences, Shanghai, 201203, P. R. China
ABSTRACT:The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other. In addition, alkaline-pH-MS/MS showed identification capacity for a higher proportion of peptides with negative grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled enrichment preference toward histidine-, lysine-, methionine-, and proline-containing peptides. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS method outperformed the conventional 120 min low-pH-MS/MS method in both the identification of amino acid variants and protein groups. Therefore, we established the alkaline-pH-MS/MS method as a simple, competitive, alternative method to low-pH-MS/MS for global proteomic analysis.
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Biochimica et biophysica acta. BioenergeticsBioenergetics. 2023. Niedzwiedzki, Dariusz M et al.
Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA
ABSTRACT:Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77K revealed two emission bands at 690nm and 725nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725nm emission band is associated with low energy Chls a with absorption band clearly resolved at 710nm at 77K. In summary, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.
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The Plant Cell2023. Kim, Tae-Wuk et al.
Carnegie Inst Sci, Dept Plant Biol, Stanford, CA 94305 USA
ABSTRACT:Combining TurboID-mediated proximity labeling with quantitative phosphoproteomics identifies BIN2 signaling components including kinase substrates in vivo, revealing cellular functions of BIN2.Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.
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Molecular Immunology2023. Wang, Ye et al.
Chinese Acad Med Sci, Peking Union Med Coll Hosp, Peking Union Med Coll, Dept Allergy, 1 Shuaifuyuan, Wangfujing 100730, Beijing, Peoples R China; Affiliated Canc Hosp Nanjing Med Univ, Jiangsu Canc Hosp, Jiangsu Inst Canc Res, Dept Pharm, 42,Baiziting Rd, Nanjing 210029, Jiangsu, Peoples R China; Childrens Hosp Nanjing Med Univ, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210008, Jiangsu, Peoples R China
ABSTRACT:Background: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the mo-lecular and immunochemical properties of this novel allergen.Methods: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods.Results: The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other ho-mologous allergens, which was a unique alpha beta alpha sandwich structure containing 4 alpha-helices and 6 antiparallel beta-sheets, encompassing 4 conserved CAP domain. Conclusion: The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.
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Toxins2023. Avella et al.
CIBIO, BIOPOLIS Program Genom Biodivers & Land Planning, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Ctr Invest Biodiversidade & Recursos Genet, InBIO Lab Associado, CIBIO, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Fac Ciencias, Dept Biol, P-4099002 Porto, Portugal
ABSTRACT:European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V. seoanei from 20 localities across the species' Iberian distribution. We used a pool of all individual venoms to generate a V. seoanei venom reference proteome, produced SDS-PAGE profiles of all venom samples, and visualised patterns of variation using NMDS. By applying linear regression, we then assessed presence and nature of venom variation between localities, and investigated the effect of 14 predictors (biological, eco-geographic, genetic) on its occurrence. The venom comprised at least 12 different toxin families, of which five (i.e., PLA(2), svSP, DI, snaclec, svMP) accounted for about 75% of the whole proteome. The comparative analyses of the SDS-PAGE venom profiles showed them to be remarkably similar across the sampled localities, suggesting low geographic variability. The regression analyses suggested significant effects of biological and habitat predictors on the little variation we detected across the analysed V. seoanei venoms. Other factors were also significantly associated with the presence/absence of individual bands in the SDS-PAGE profiles. The low levels of venom variability we detected within V. seoanei might be the result of a recent population expansion, or of processes other than directional positive selection.
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APPLIED SCIENCES-BASEL2023. Xiang Zhang et al.
Shandong Univ Technol, Sch Comp Sci & Technol, Zibo 255049, Peoples R China
ABSTRACT:Although data-independent acquisition (DIA) has the ability to identify and quantify all peptides in a sample, highly complex mixed mass spectra present difficulties for accurate peptide and protein identification. Additionally, the correspondence between the precursor and its fragments is broken, making it challenging to perform peptide identification directly using conventional DDA search engines. In this paper, we propose a cosine-similarity-based deconvolution method: CorrDIA. This is achieved by reconstructing the correspondence between precursor and fragment ions based on the consistency of extracted ion chromatograms (XICs). A deisotope peak cluster operation is added and centered on the MS/MS spectrum to improve the accuracy of spectrum interpretation and increase the number of identified peptides. The resulting MS/MS spectra can be identified using any data-dependent acquisition (DDA) sequencing software. The experimental results demonstrate that the number of peptide results increased by 12 percent and 21 percent respectively, and the repetition rate decreased by 12 percent. This reduces mass spectra complexity and difficulties in mass spectra analysis without the need for any mass spectra libraries.
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