pFind Studio: a computational solution for mass spectrometry-based proteomics
2020
Journal of experimental botany2020. Kocab, O et al.
Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
ABSTRACT:Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. x Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.
Use: pNovo
Journal of experimental botany2020. Kocab, O et al.
Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
ABSTRACT:Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. x Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.
Use: pNovo
Analytical Chemistry2020. Capriotti, AL et al.
Sapienza Univ Roma, Dept Chem, I-00185 Rome, Italy.
ABSTRACT:Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.
Use: pNovo
Talanta2020. Cerrato, A et al.
Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT:Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose. First, they were employed as inclusion lists for a suspect screening mass-spectrometric analysis, overcoming the limits of data dependent acquisition mode and allowing the fragmentation of such low-abundance substances. Moreover, the databases were implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, for the creation of a data processing workflow specifically dedicated to short peptide tentative identification. For this purpose, a detailed study of short peptide fragmentation pathways was carried out for the first time. The proposed method was applied to the study of short peptide sequences in enriched urine samples and led to the tentative identification more than 200 short natural and modified short peptides, the highest number ever reported.
Use: pNovo
FASEB JOURNAL2020. Biggart, MGS et al.
St Georges University Of London
ABSTRACT:The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI-1 (NHBE-BMI1) and cell-lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air-liquid interface with PBS (100l) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides < 10kDa, were subsequently isolated and analysed using the Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovoDe novosequencing tool. Our preliminary results identified 4765 unique peptides (NHBE-BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline-alanine rich N-terminal region and a cationic C-terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescentPseudomonas aeruginosaover 18 hours at 37C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.631.34 M (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine-proline rich N-terminal sequence or the cationic C-terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.062.27% and 35.289.45% respectively compared to the full-length peptide; 99.990.001% (all normalised to untreated bacterial luminescence, n=4, p<0.0001, SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone-derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.
Use: pNovo
Nature Communications2020. Fang, P et al.
Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.
ABSTRACT:Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt's lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence. Comprehensive quantitative profiling of intact glycopeptides remains technically challenging. To address this, the authors here develop an integrated quantitative glycoproteomic workflow, including optimized sample preparation, multiplexed quantification and a dedicated data processing tool.
Use: pGlyco; pParse
Analytical chemistry2020. Zhu, H et al.
Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT:Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of alpha 2,3/alpha 2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of alpha 2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of alpha 2,6-sailoglycopeptide isomers. 'With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.
Use: pParse; pGlyco
Rapid Communications in Mass Spectrometry2020. Jeong, HK et al.
Korea Basic Sci Inst, Res Ctr Bioconvergence Anal, 162 Yeongudanji Ro, Cheongju 28119, South Korea.
ABSTRACT:Rationale Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods The relative intensities of the Y1F, Y2F, Y3F, and Y4F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation. The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2F and B3SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. Results In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. Conclusions We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine.
Use: pGlyco
Cell host & microbe2020. Zhao, P et al.
Univ Georgia, Dept Biochem & Mol Biol, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA.
ABSTRACT:The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.
Use: pGlyco
Engineering (Beijing, China)2020. Sun, Zeyu et al.
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310011, China
ABSTRACT:The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2. Additional post-translational modification (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy (cryo-EM) structures. The PTM and glycan maps of HCoV-19 S and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines. 2020 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company.
Use: pGlyco