pFind Studio: a computational solution for mass spectrometry-based proteomics
2022
Analytical Chemistry2022. An, YX et al.
Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Liaoning, Peoples R China
ABSTRACT:Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal. In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score
Use: pLink
Journal of Biological Chemistry2022. Huang, CA et al.
Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Renji Hosp Res Ctr, Renji Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Inst, Minist Educ, Key Lab Genet Dev & Neuropsychiat Disorders, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China
ABSTRACT:Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD(+) synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including alpha-synuclein, Tau (K19), amyloid beta, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.
Use: pLink
International Journal of Mass Spectrometry2022. Zuo, Mei-Qing et al.
Natl Inst Biol Sci, Beijing, Peoples R China
ABSTRACT:Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not. The most frequent side-chain NLs involve amino acids C, S, T, D, E, N, Q, and R. Although NL of CO2 is common for all peptides, it is markedly enhanced in D/E-containing peptides. Long peptides and D/E-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, 6-, c-, x-, y-, z -ions) than those produced from 1- ([M -H]-) and 2- ([M -2H]2-) precursors. Based on the above findings, we predict that under negative-ion HCD, D/E-rich peptides should be difficult to identify and that choosing a protease to generate peptides containing fewer D/E residues will improve identification of highly acidic proteins.(c) 2022 Elsevier B.V. All rights reserved.
Use: pLink
Journal of Cell Biology2022. Jiang, QQ et al.
Univ Penn, Penn Ctr Genome Integr, Basser Ctr BRCA, Perelman Sch Med,Dept Canc Biol, Philadelphia, PA 19104 USA; Univ Leeds, Fac Biol Sci, Astbury Ctr Struct Mol Biol, Sch Mol & Cellular Biol, Leeds, W Yorkshire, England
ABSTRACT:The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.
Use: pLink
International Journal of Mass Spectrometry2022. Zuo, Mei-Qing et al.
Natl Inst Biol Sci, Beijing, Peoples R China
ABSTRACT:Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not. The most frequent side-chain NLs involve amino acids C, S, T, D, E, N, Q, and R. Although NL of CO2 is common for all peptides, it is markedly enhanced in D/E-containing peptides. Long peptides and D/E-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, 6-, c-, x-, y-, z -ions) than those produced from 1- ([M -H]-) and 2- ([M -2H]2-) precursors. Based on the above findings, we predict that under negative-ion HCD, D/E-rich peptides should be difficult to identify and that choosing a protease to generate peptides containing fewer D/E residues will improve identification of highly acidic proteins.(c) 2022 Elsevier B.V. All rights reserved.
Use: pLink
Analytical Chemistry2022. Ruwolt, M et al.
Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany
ABSTRACT:Cross-linking mass spectrometry (XL-MS) is a powerful method for theinvestigation of protein-protein interactions (PPI) from highly complex samples. XL-MScombined with tandem mass tag (TMT) labeling holds the promise of large-scale PPIquantification. However, a robust and efficient TMT-based XL-MS quantificationmethod has not yet been established due to the lack of a benchmarking dataset andthorough evaluation of various MS parameters. To tackle these limitations, we generate atwo-interactome dataset by spiking in TMT-labeled cross-linkedEscherichia colilysateinto TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Usingthis benchmarking dataset, we assess the efficacy of cross-link identification and accuracyof cross-link quantification using different MS acquisition strategies. For identification,we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higherenergy collisional dissociation (HCD) energies for TMT-labeled cross-links. Weobserved a need for notably higher fragmentation energies compared to unlabeledcross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursorselection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vastnumber of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexedquantitative PPI characterization from complex biological systems.
Use: pLink
ACS omega2022. Choong, WK et al.
Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan
ABSTRACT:Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking. Therefore, we propose a framework of three bottom-uplevels, peptide-spectrum match, peptide, and variant event levels,that consists of rigorous 11-aspect examinations from the MSperspective to further confirm the reliability of variant events. As aproof of concept and showing feasibility, we demonstrate 11 examinations on the identified variant peptides from an HEK293 cellline data set, where various database search strategies were applied to maximize the number of identified variant PSMs with an FDR<1% for postexaminations. The results showed that only FDR criterion is insufficient to validate identified variant peptides and the11 postexaminations can reveal low-confidence variant events detected by shotgun proteomics experiments. Therefore, we suggestthat postexaminations of identified variant events based on the proposed framework are necessary for proteogenomics studies
Use: pNovo
Molecular immunology2022. Xu, ZQ et al.
Nanjing Med Univ, Dept Pharm, Jiangsu Canc Hosp, Affiliated Canc Hosp,Jiangsu Inst Canc Res, 42 Baiziting Rd, Nanjing 210029, Peoples R China; Nanjing Med Univ, Childrens Hosp, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210000, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Allergy, 1 Shuaifuyuan, Beijing 100730, Peoples R China
ABSTRACT:Background: Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach.Methods: The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning. Both the recombinant and natural protein molecules were purified and characterized. The allergenicity was further analyzed by enzyme linked immunosorbent assay, immunoblot, and basophil activation test using sera from cockroach allergic patients.Results: A novel allergen belonging to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was firstly identified in the American cockroach and named as Per a 13. The cDNA of this allergen is 1255 base pairs in length and contains an open reading frame of 999 base pairs, encoding 332 amino acids. The purified Per a 13 was fully characterized and assessed to react with IgEs from 49.3 % of cockroach allergic patients, and patients with allergic rhinitis were more sensitized to it. Moreover, the allergenicity was further confirmed by immunoblot and basophil activation test.Conclusions: We firstly identified GAPDH (Per a 13) in the American cockroach, which is a novel type of inhalant allergen derived from animal species. These findings could be useful in developing novel diagnostic and therapeutic strategies for cockroach allergy.
Use: pNovo
Molecular Immunology2022. Ling, XJ et al.
Gannan Med Univ, Dept Blood Transfus, Affiliated Hosp 1, Ganzhou 341000, Jiangxi, Peoples R China; Nanjing Med Univ, Jiangsu Canc Hosp, Dept Pharm, Nanjing 210009, Peoples R China; Peking Union Med Coll Hosp, Chinese Acad Med Sci & Peking Union Med Coll, State Key Lab Complex Severe & Rare Dis, Allergy Dept, Beijing 100730, Peoples R China; Nanjing Med Univ, Affiliated Canc Hosp, Nanjing 210009, Peoples R China; Nanjing Med Univ, Jiangsu Inst Canc Res, Nanjing 210009, Peoples R China
ABSTRACT:Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteineprotease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen specific immunotherapy.
Use: pNovo
ACS omega2022. Choong, WK et al.
Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan
ABSTRACT:Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking. Therefore, we propose a framework of three bottom-uplevels, peptide-spectrum match, peptide, and variant event levels,that consists of rigorous 11-aspect examinations from the MSperspective to further confirm the reliability of variant events. As aproof of concept and showing feasibility, we demonstrate 11 examinations on the identified variant peptides from an HEK293 cellline data set, where various database search strategies were applied to maximize the number of identified variant PSMs with an FDR<1% for postexaminations. The results showed that only FDR criterion is insufficient to validate identified variant peptides and the11 postexaminations can reveal low-confidence variant events detected by shotgun proteomics experiments. Therefore, we suggestthat postexaminations of identified variant events based on the proposed framework are necessary for proteogenomics studies
Use: pNovo