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1.
Se Pu ; 39(10): 1045-1054, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505426

RESUMO

Protein glycosylation is one of the most important post-translational modifications (PTMs). The glycosylation is crucial in a variety of physiological and pathological processes that include protein stability, intracellular and intercellular signal transduction, hormone activation or inactivation, and immune regulation. Protein glycosylation is generated by complex biosynthetic pathways comprising hundreds of glycosyltransferases, glycosidases, transcriptional factors, transporters, and protein backbones. Abnormal protein glycosylation is closely associated with the occurrence and development of diseases. Many disease biomarkers in clinical screening are glycoproteins (alfa fetoprotein for liver cancer, carbohydrate antigen 125 for ovarian cancer, carcinoembryonic antigen for colon cancer, prostate-specific antigen for prostate cancer, etc.), and glycan antigens (carbohydrate antigen 19-9 for gastrointestinal cancer and pancreatic cancer, etc.). Glycoproteomics research and technological developments are important to elucidate the mechanism of protein glycosylation in vivo. Mass spectrometry (MS)-based proteomics provides an excellent approach for the comprehensive analysis of proteins and their modifications. In bottom-up proteomics, glycoproteomic analysis is more difficult than other PTMs because intact glycopeptides have diverse peptide backbones and glycan chains, relatively low abundance and ionization efficiency, and pronounced heterogeneity. In recent years, glycoproteomic methodologies such as intact glycopeptide enrichment methods, MS fragmentation and acquisition approaches, MS data interpretation tools and software, and quantification strategies have been appreciably improved. These methodologies have driven in-depth glycoproteomics research. This review focuses on the recent advances in MS-based glycoproteomics. New enrichment methods and spectral interpretation approaches of intact N- and O-glycopeptides are discussed. Their applications in answering various questions in complex biological systems are also considered. The new enrichment methods for intact glycopeptides are mostly based on existing principles. Some properties of the materials, such as hydrophilicity and electrophilicity, have been optimized to improve the enrichment performance. For example, dual-functional Ti(IV)-IMAC materials have been used for the separation of glycopeptides and phosphopeptides. Considering the clinical applications, some glycoproteomics methods integrate enrichment processing into automated workflows to reduce errors caused by manual operations and to increase the experimental reproducibility and efficiency. For example, an automated glycopeptide enrichment method consisting of a liquid chromatograph equipped with a hydrophilic interaction chromatography column has been shown capable of highly reproducible analyses of site-specific glycopeptides in complex biological samples. These methods are more suitable for the discovery of newly glycosylation-related biomarkers as well as for the physiopathological studies of human diseases. With the optimization of glycopeptide enrichment methods and the innovation of MS technologies in the past decade, MS analysis of intact glycopeptides has begun to yield a wealth of glycopeptide fragment ions and plentiful high-quality MS data. This review introduces several effective fragmentation methods for intact glycopeptides. These include collision-induced dissociation, high-energy collision dissociation, electron capture dissociation, electron-transfer dissociation, and electron-transfer/higher-energy collision dissociation. Automated analysis of MS data of intact N- and O-glycopeptides requires interpretation approaches and corresponding software tools with high sensitivity and reliability. Finally, we highlight the utility of several spectral interpretation approaches and their corresponding popular search software, including ArMone, Byonic, GPQuest, pGlyco, O-search, MSFragger-Glyco, and O-Pair Search. In addition, MS data acquisition modes, such as data-dependent acquisition, data-independent acquisition, multiple reaction monitoring technology, and parallel reaction monitoring technology, have great application prospects in glycoproteomics research. With the improvements in enrichment methods, MS technologies, and spectral interpretation approaches for intact N- and O-glycopeptides, comprehensive and systematic glycoproteomics analysis has tremendously expanded the knowledge of protein glycosylation. These glycoproteomic technologies have a wide range of applications that include exploring the molecular mechanisms of protein glycosylation and discovering the new biomarkers of human diseases.


Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Humanos , Masculino , Proteômica , Reprodutibilidade dos Testes
2.
Se Pu ; 39(10): 1086-1093, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505430

RESUMO

Many secreted proteins, including cytokines, growth factors and hormones, are crucial in processes like intercellular signaling. Dynamic changes in secreted proteins usually reflect the growth and pathological state of the cells. Many drug targets are secretory proteins. The proteins are also important biomarkers. Conditioned cell culture media are important samples for secretory proteomic studies. Biomass spectrometry-based proteomic analysis enables the systematic study of secretory proteins. The main problem in analyzing secretory proteins in conditioned culture media is the low concentration of these proteins and the presence of serum, amino acids, and additives in culture media that may interfere with the protein analysis. Conventional secretory proteome analysis uses serum-free cell culture to reduce sample complexity, and typically involves protein concentration, purification, and desalting using ultrafiltration, dialysis, lyophilization, and trichloroacetic acid (TCA) or acetone precipitation, followed by enzymatic digestion and mass spectrometry analysis. This analytical process does not allow specific enrichment of secreted proteins. Thus, only a few secreted proteins can be identified. In addition, prolonged serum-free incubation of cells also tends to lead to unexpected changes in their activity status. A bioorthogonal-based enrichment approach can effectively avoid this problem. In recent years, unnatural sugars containing bio-orthologous groups, such as azide groups, have been used to metabolically label glycosylated proteins, enabling cellular imaging or selective enrichment of glycoproteins and their use for proteomic analysis. The strategy is a two-step process. First, azide-based sugar analogues are added to the cell culture medium and introduced to glycoproteins via the intracellular glycan biosynthesis pathway. Second, they are specifically covalently labeled with imaging probes or affinity probes via click chemistry. Since secreted proteins are usually glycoproteins, this glycolytic labeling has been used to label and enrich secreted proteins. N-Azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz), and N-azidoacetylmannosamine (ManNAz) are classical azide-based sugar analogues. Their effects on cytoplasmic membrane proteins have been compared. However, only ManNAz has been used for metabolic labeling of secreted proteins. No other glyco-analogues that label secreted proteins have been reported. Here, the bio-orthogonal chemical biology technology achieved highly selective labeling and enriched secreted proteins. In combination with click chemistry, different sugar analogues were evaluated for metabolic labeling of secreted proteins. HeLa cells were metabolically labeled by ManNAz, GalNAz, and GlcNAz (the three most commonly used commercial sugar analogues). These glycolytic markers can selectively label specific types of glycosylation. For example, ManNAz, an analogue of the biosynthetic precursor of sialic acid, N-acetylmannosamine (ManNAc), can label sialylated N- or O-glycoproteins. GalNAz, an analogue of N-acetylgalactosamine (GalNAc), can replace GalNAc as a core residue of mucin-type O-glycans and thus label O-glycoproteins. In addition, the intracellular metabolic intermediate of GalNAz (pyrophosphate) UDP-GalNAz can be interconverted with UDP-GlcNAz catalyzed by UDP-galactose-4-differential isomerase (GALE) and thus can also label N-glycoproteins and O-GlcNAc glycoproteins instead of GlcNAc. The GlcNAz analogue is commonly used to label nuclear and cytoplasmic glycoproteins with β-O-GlcNAc residues, but can also label N-glycoproteins with mucin-type O-glycoproteins by converting GALE to GalNAz, followed by enrichment using a biotin-alkynyl probe. Label-free quantitative proteomic analysis was performed to evaluate their labeling efficiency. ManNAz-based secretory protein labeling identified 282 secretory proteins, 224 plasma membrane proteins, and 846 N-glycosites. Compared with GalNAz and GlcNAz, the enrichment of secreted proteins was increased 130% and 67.2%, respectively, and the enrichment of plasma membrane proteins was increased 273.3% and 148.7%, respectively. This study provides a useful comparative analysis and new strategies for highly selective enrichment and systematic secretome analysis.


Assuntos
Proteômica , Açúcares , Glicoproteínas , Glicosilação , Células HeLa , Humanos
3.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3934-3942, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34472270

RESUMO

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 µmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.


Assuntos
Peróxido de Hidrogênio , Fosfatidilinositol 3-Quinases , Animais , Medicamentos de Ervas Chinesas , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
4.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3943-3948, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34472271

RESUMO

The study aims to investigate the effect of the compatibility of paeonol and paeoniflorin(hereinafter referred to as the compatibility) on the expression of myocardial proteins in rats with myocardial ischemia injury and explore the underlying mechanism of the compatibility against myocardial ischemia injury. First, the acute myocardial infarction rat model was established by ligation of the anterior descending branch of the left coronary artery. The model rats were given(ig) paeonol and paeoniflorin. Then protein samples were collected from rat cardiac tissue and quantified by tandem mass tags(TMT) to explore the differential proteins after drug intervention. The experimental results showed that differential proteins mainly involved phagocytosis engulfment, extracellular space, and antigen binding, as well as Kyoto encyclopedia of genes and genomes(KEGG) pathways of complement and coagulation cascades, syste-mic lupus erythematosus, and ribosome. In this study, the target proteins and related signaling pathways identified by differential proteomics may be the biological basis of the compatibility against myocardial ischemia injury in rats.


Assuntos
Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Acetofenonas , Animais , Glucosídeos , Monoterpenos , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/genética , Proteômica , Ratos , Ratos Sprague-Dawley
5.
BMC Genomics ; 22(1): 648, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493209

RESUMO

BACKGROUND: Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling. RESULTS: In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential. CONCLUSIONS: Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.


Assuntos
Bacillus cereus , Proteoma , Anaerobiose , Oxirredução , Proteoma/metabolismo , Proteômica , Compostos de Sulfidrila
6.
World J Gastroenterol ; 27(31): 5171-5180, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34497442

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) represents a challenging pathology with very poor outcomes and is increasing in incidence within the general population. The majority of patients are diagnosed incidentally with insidious symptoms and hence present late in the disease process. This significantly affects patient outcomes: the only cure is surgical resection but only up to 20% of patients present with resectable disease at the time of clinical presentation. The use of "omic" technology is expanding rapidly in the field of personalised medicine - using genomic, proteomic and metabolomic approaches allows researchers and clinicians to delve deep into the core molecular processes of this difficult disease. This review gives an overview of the current findings in PDAC using these "omic" approaches and summarises useful markers in aiding clinicians treating PDAC. Future strategies incorporating these findings and potential application of these methods are presented in this review article.


Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/genética , Humanos , Metabolômica , Neoplasias Pancreáticas/genética , Proteômica
7.
Mater Sci Eng C Mater Biol Appl ; 128: 112289, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474840

RESUMO

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH2-terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation.


Assuntos
Proteoma , Titânio , Diamante , Humanos , Osteoblastos , Proteômica , Propriedades de Superfície
8.
Mater Sci Eng C Mater Biol Appl ; 128: 112312, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474863

RESUMO

Meniscus is a fibrocartilage composite tissue with three different microstructual zones, inner fibrocartilage, middle transitional, and outer fibrous zone. We hypothesized that decellularized meniscus extracellular matrix (DMECM) would have different characteristics according to zone of origin. We aimed to compare zone-specific DMECM in terms of biochemical characteristics and cellular interactions associated with tissue engineering. Micronized DMECM was fabricated from porcine meniscus divided into three microstructural zones. Characterization of DMECM was done by biochemical and proteomic analysis. Inner DMECM showed the highest glycosaminoglycan content, while middle DMECM showed the highest collagen content among groups. Proteomic analysis showed significant differences among DMECM groups. Inner DMECM showed better adhesion and migration potential to meniscus cells compared to other groups. DMECM resulted in expression of zone-specific differentiation markers when co-cultured with synovial mesenchymal stem cells (SMSCs). SMSCs combined with inner DMECM showed the highest glycosaminoglycan in vivo. Outer DMECM constructs, on the other hand, showed more fibrous tissue features, while middle DMECM constructs showed both inner and outer zone characteristics. In conclusion, DMECM showed different characteristics according to microstructural zones, and such material may be useful for zone-specific tissue engineering of meniscus.


Assuntos
Menisco , Proteômica , Animais , Matriz Extracelular , Meniscos Tibiais , Suínos , Engenharia Tecidual
9.
Urologiia ; (4): 126-131, 2021 Sep.
Artigo em Russo | MEDLINE | ID: mdl-34486285

RESUMO

The formation and development theories of bacterial inflammation in organs and tissues have been studied in detail and confirmed by experimental and clinical data. However, the development of inflammation in each organ has its characteristics associated with its structures and functions. This fully applies to acute pyelonephritis. The peculiar structure of uropathogens, their virulence, the reaction of the host organism in response to bacterial invasion, namely, factors of innate and acquired immunity, are analyzed in detail in the review. It reflects both the basic mechanisms of development of acute pyelonephritis and proteomic and genetic factors involved during inflammatory lesions.


Assuntos
Proteômica , Pielonefrite , Bactérias , Humanos
10.
Microbiome ; 9(1): 182, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34479645

RESUMO

BACKGROUND: Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown. RESULTS: We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR-Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction-modification systems and dormancy-inducing toxin-antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication. CONCLUSIONS: This study provides new insights into phage-bacteria interplay in intracellular environments of a deep-sea vent snail. Video Abstract.


Assuntos
Bacteriófagos , Animais , Bactérias/genética , Bacteriófagos/genética , Genômica , Proteômica , Caramujos , Transcriptoma/genética
11.
Nutrients ; 13(8)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34444642

RESUMO

Fatty acids play a significant role in maintaining cellular and DNA protection and we previously found an inverse relationship between blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and DNA damage. The aim of this study was to explore differences in proteomic profiles, for 117 pro-inflammatory proteins, in two previously defined groups of individuals with different DNA damage and EPA and DHA levels. Healthy children and adolescents (n = 140) aged 9 to 13 years old in an urban area of Brazil were divided by k-means cluster test into two clusters of DNA damage (tail intensity) using the comet assay (cluster 1 = 5.9% ± 1.2 and cluster 2 = 13.8% ± 3.1) in our previous study. The cluster with higher DNA damage and lower levels of DHA (6.2 ± 1.6 mg/dL; 5.4 ± 1.3 mg/dL, p = 0.003) and EPA (0.6 ± 0.2 mg/dL; 0.5 ± 0.1 mg/dL, p < 0.001) presented increased expression of the proteins CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB, which are involved in pro-inflammatory pathways. Our findings support the hypothesis that low levels of n-3 long-chain PUFA may have a less protective role against DNA damage through expression of pro-inflammatory proteins, such as CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB.


Assuntos
Dano ao DNA , Ácidos Docosa-Hexaenoicos/sangue , Ácido Eicosapentaenoico/sangue , Ácidos Graxos Ômega-3/sangue , Adolescente , Brasil , Criança , Classe I de Fosfatidilinositol 3-Quinases/sangue , Classe Ia de Fosfatidilinositol 3-Quinase/sangue , Estudos Transversais , Ciclina C/sangue , Quinase 8 Dependente de Ciclina/sangue , Feminino , Humanos , Hidrolases/sangue , Inflamação/metabolismo , Masculino , Proteína Quinase C beta/sangue , Proteômica
12.
Anal Chem ; 93(33): 11415-11423, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34375078

RESUMO

Targeted, untargeted, and data-independent acquisition (DIA) metabolomics workflows are often hampered by ambiguous identification based on either MS1 information alone or relatively few MS2 fragment ions. While DIA methods have been popularized in proteomics, it is less clear whether they are suitable for metabolomics workflows due to their large precursor isolation windows and complex coisolation patterns. Here, we quantitatively investigate the conditions necessary for unique metabolite detection in complex backgrounds using precursor and fragment ion mass-to-charge (m/z) separation, comparing three benchmarked mass spectrometry (MS) methods [MS1, MRM (multiple reaction monitoring), and DIA]. Our simulations show that DIA outperformed MS1-only and MRM-based methods with regards to specificity by factors of ∼2.8-fold and ∼1.8-fold, respectively. Additionally, we show that our results are not dependent on the number of transitions used or the complexity of the background matrix. Finally, we show that collision energy is an important factor in unambiguous detection and that a single collision energy setting per compound cannot achieve optimal pairwise differentiation of compounds. Our analysis demonstrates the power of using both high-resolution precursor and high-resolution fragment ion m/z for unambiguous compound detection. This work also establishes DIA as an emerging MS acquisition method with high selectivity for metabolomics, outperforming both data-dependent acquisition (DDA) and MRM with regards to unique compound identification potential.


Assuntos
Metabolômica , Proteômica , Íons , Espectrometria de Massas , Fluxo de Trabalho
13.
Anal Chem ; 93(33): 11442-11450, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34375526

RESUMO

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas-phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) bovine serum albumin (BSA), (ii) Escherichia coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1623 unique cross-links for nuclear lysates, corresponding to 1088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving three-dimensional (3D) structures of single proteins but also for performing system-wide protein interaction studies.


Assuntos
Escherichia coli , Proteômica , Reagentes para Ligações Cruzadas , Células HEK293 , Humanos , Íons , Espectrometria de Massas , Soroalbumina Bovina
14.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443345

RESUMO

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.


Assuntos
Algoritmos , Glicopeptídeos/análise , Glicoproteínas/análise , COVID-19/metabolismo , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Proteômica/métodos , Proteômica/normas , SARS-CoV-2/metabolismo , Software , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/química , Espectrometria de Massas em Tandem/métodos , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/química
15.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360786

RESUMO

Since the emergence of high-throughput proteomic techniques and advances in clinical technologies, there has been a steady rise in the number of cancer-associated diagnostic, prognostic, and predictive biomarkers being identified and translated into clinical use. The characterisation of biofluids has become a core objective for many proteomic researchers in order to detect disease-associated protein biomarkers in a minimally invasive manner. The proteomes of biofluids, including serum, saliva, cerebrospinal fluid, and urine, are highly dynamic with protein abundance fluctuating depending on the physiological and/or pathophysiological context. Improvements in mass-spectrometric technologies have facilitated the in-depth characterisation of biofluid proteomes which are now considered hosts of a wide array of clinically relevant biomarkers. Promising efforts are being made in the field of biomarker diagnostics for haematologic malignancies. Several serum and urine-based biomarkers such as free light chains, ß-microglobulin, and lactate dehydrogenase are quantified as part of the clinical assessment of haematological malignancies. However, novel, minimally invasive proteomic markers are required to aid diagnosis and prognosis and to monitor therapeutic response and minimal residual disease. This review focuses on biofluids as a promising source of proteomic biomarkers in haematologic malignancies and a key component of future diagnostic, prognostic, and disease-monitoring applications.


Assuntos
Biomarcadores Tumorais/metabolismo , Líquidos Corporais/metabolismo , Neoplasias Hematológicas , Proteínas de Neoplasias/metabolismo , Proteômica , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/metabolismo , Humanos
16.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360799

RESUMO

Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological disease, causing motor and cognitive dysfunction and dementia. iNPH and Alzheimer's disease (AD) share similar molecular characteristics, including amyloid deposition, t-tau and p-tau dysregulation; however, the disease is under-diagnosed and under-treated. The aim was to identify a panel of sphingolipids and proteins in CSF to diagnose iNPH at onset compared to aged subjects with cognitive integrity (C) and AD patients by adopting multiple reaction monitoring mass spectrometry (MRM-MS) for sphingolipid quantitative assessment and advanced high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic analysis. The results indicated that iNPH are characterized by an increase in very long chains Cer C22:0, Cer C24:0 and Cer C24:1 and of acute-phase proteins, immunoglobulins and complement component fragments. Proteins involved in synaptic signaling, axogenesis, including BACE1, APP, SEZ6L and SEZ6L2; secretory proteins (CHGA, SCG3 and VGF); glycosylation proteins (POMGNT1 and DAG1); and proteins involved in lipid metabolism (APOH and LCAT) were statistically lower in iNPH. In conclusion, at the disease onset, several factors contribute to maintaining cell homeostasis, and the protective role of very long chains sphingolipids counteract overexpression of amyloidogenic and neurotoxic proteins. Monitoring specific very long chain Cers will improve the early diagnosis and can promote patient follow-up.


Assuntos
Hidrocefalia de Pressão Normal/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteômica , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esfingolipídeos/líquido cefalorraquidiano
17.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360826

RESUMO

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Eritrócitos/metabolismo , Glicômica , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicosilação , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360830

RESUMO

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas do Olho/metabolismo , Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Transporte Proteico , Animais , Cromatografia Líquida , Cílios , Proteínas do Olho/genética , Imunoprecipitação , Camundongos , Camundongos Knockout , Ligação Proteica , Proteômica , Ratos , Sinapses , Espectrometria de Massas em Tandem
19.
Nat Commun ; 12(1): 5015, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408139

RESUMO

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/química , Proteômica
20.
Microb Pathog ; 159: 105150, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34425197

RESUMO

Salmonella enterica serovar Typhi (S. Typhi), a causative agent of typhoid fever, is a Gram-negative, human-restricted pathogen that causes significant morbidity and mortality, particularly in developing countries. The currently available typhoid vaccines are not recommended to children below six years of age and have poor long-term efficacy. Due to these limitations and the emerging threat of multidrug-resistance (MDR) strains, the development of a new vaccine is urgently needed. The present study aims to design a multiepitope-based subunit vaccine (MESV) against MDR S. Typhi str. CT18 using a computational-based approach comprising subtractive proteomics and immunoinformatics. Firstly, we investigated the proteome of S. Typhi str. CT18 using subtractive proteomics and identified twelve essential, virulent, host non-homologous, and antigenic outer membrane proteins (OMPs) as potential vaccine candidates with low transmembrane helices (≤1) and molecular weight (≤110 kDa). The OMPs were mapped for cytotoxic T lymphocyte(CTL) epitopes, helper T lymphocyte (HTL) epitopes, and linear B lymphocyte (LBL) epitopes using various immunoinformatics tools and servers. A total of 6, 12, and 11 CTL, HTL, and LBL epitopes were shortlisted, respectively, based on their immunogenicity, antigenicity, allergenicity, toxicity, and hydropathicity potential. Four MESV constructs (MESVCs), MESVC-1, MESVC-2, MESVC-3, and MESVC-4, were designed by linking the CTL, HTL, and LBL epitopes with immune-modulating adjuvants, linkers, and PADRE (Pan HLA DR-binding epitope) sequences. The MESVCs were evaluated for their physicochemical properties, allergenicity, antigenicity, toxicity, and solubility potential to ensure their safety and immunogenic behavior. Secondary and tertiary structures of shortlisted MESVCs (MESVC-1, MESVC-3, and MESVC-4) were predicted, modeled, refined, validated, and then docked with various MHC I, MHC II, and TLR4/MD2 complex. Molecular dynamics (MD) simulation of the final selected MESVC-4 with TLR4/MD2 complex confirms its binding affinity and stability. Codon optimization and in silico cloning verified the translation efficiency and successful expression of MESVC-4 in E. coli str. K12. Finally, the efficiency of MESVC-4 to trigger an effective immune response was assessed by an in silico immune simulation. In conclusion, our findings show that the designed MESVC-4 can elicit humoral and cellular immune responses, implying that it may be used for prophylactic or therapeutic purposes. Therefore, it should be subjected to further experimental validations.


Assuntos
Proteômica , Salmonella typhi , Criança , Biologia Computacional , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Escherichia coli , Humanos , Simulação de Acoplamento Molecular , Salmonella typhi/genética , Vacinas de Subunidades
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