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1.
Braz. j. biol ; 83: e249424, 2023. graf
Artigo em Inglês | MEDLINE, LILACS, VETINDEX | ID: biblio-1345538

RESUMO

Abstract Hypoxia is a prominent feature of head and neck cancer. However, the oxygen element characteristics of proteins and how they adapt to hypoxia microenvironments of head and neck cancer are still unknown. Human genome sequences and proteins expressed data of head and neck cancer were retrieved from pathology atlas of Human Protein Atlas project. Then compared the oxygen and carbon element contents between proteomes of head and neck cancer and normal oral mucosa-squamous epithelial cells, genome locations, pathways, and functional dissection associated with head and neck cancer were also studied. A total of 902 differentially expressed proteins were observed where the average oxygen content is higher than that of the lowly expressed proteins in head and neck cancer proteins. Further, the average oxygen content of the up regulated proteins was 2.54% higher than other. None of their coding genes were distributed on the Y chromosome. The up regulated proteins were enriched in endocytosis, apoptosis and regulation of actin cytoskeleton. The increased oxygen contents of the highly expressed and the up regulated proteins might be caused by frequent activity of cytoskeleton and adapted to the rapid growth and fast division of the head and neck cancer cells. The oxygen usage bias and key proteins may help us to understand the mechanisms behind head and neck cancer in targeted therapy, which lays a foundation for the application of stoichioproteomics in targeted therapy and provides promise for potential treatments for head and neck cancer.


Resumo A hipóxia é uma característica proeminente do câncer de cabeça e pescoço. No entanto, as características do elemento oxigênio das proteínas e como elas se adaptam aos microambientes de hipóxia do câncer de cabeça e pescoço ainda são desconhecidas. Sequências do genoma humano e dados expressos de proteínas de câncer de cabeça e pescoço foram recuperados do atlas de patologia do projeto Human Protein Atlas. Em seguida, comparou o conteúdo do elemento de oxigênio e carbono entre proteomas de câncer de cabeça e pescoço, e células epiteliais escamosas da mucosa oral normal, localizações do genoma, vias e dissecção funcional associada ao câncer de cabeça e pescoço também foram estudadas. Um total de 902 proteínas expressas diferencialmente foi observado onde o conteúdo médio de oxigênio é maior do que as proteínas expressas de forma humilde em proteínas de câncer de cabeça e pescoço. Além disso, o conteúdo médio de oxigênio das proteínas reguladas positivamente foi 2,54% maior do que das outras. Nenhum de seus genes codificadores foi distribuído no cromossomo Y. As proteínas reguladas positivamente foram enriquecidas em endocitose, apoptose e regulação do citoesqueleto de actina. O conteúdo aumentado de oxigênio das proteínas altamente expressas e reguladas pode ser causado pela atividade frequente do citoesqueleto e adaptado ao rápido crescimento e divisão das células cancerosas de cabeça e pescoço. O viés do uso de oxigênio e as proteínas-chave podem nos ajudar a entender os mecanismos por trás do câncer de cabeça e pescoço na terapia direcionada, o que estabelece uma base para a aplicação da estequioproteômica na terapia direcionada e oferece uma promessa para potenciais tratamentos para o câncer de cabeça e pescoço.


Assuntos
Humanos , Neoplasias de Cabeça e Pescoço/genética , Oxigênio , Carbono , Proteoma/genética , Microambiente Tumoral
2.
Life Sci Space Res (Amst) ; 33: 7-12, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35491032

RESUMO

The interest in the role of the gravitational factor during landing after long-term space flights (SF) leads to the search for various innovative approaches to assessing the compliance of external changes observed by clinicians. The results of special research methods such as Omics technologies that may reflect physiological responses to the conditions created during landing are of great interest. Our purpose is to compare the blood plasma proteome changes associated with the trauma and endothelial dysfunction processes prior to launch and on the day of landing, as well as the groups of cosmonauts with and without the secondary hemorrhagic purpura. In our study, the concentrations of 125 plasma proteins in 18 Russian cosmonauts, measured using targeted proteomic analysis based on liquid chromatography and tandem mass spectrometry were analyzed. The results reveal the trends of 12 proteins participating in the processes that trigger hemorrhagic purpura under the effect of re-entry g-forces. Exposure to intense g-forces and return to the gravity are the key factors for external manifestations of changes in the body systems induced by a long-term stay in space microgravity. Our results may be useful for further research to experts in gravitational physiology, aviation and space medicine.


Assuntos
Astronautas , Púrpura , Humanos , Plasma/química , Proteoma/análise , Proteômica
3.
Oxid Med Cell Longev ; 2022: 3725056, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35502212

RESUMO

Since aging has been associated with increased production of inflammatory biomarkers, the ability to monitor older adults repeatedly is highly desirable, and saliva is an interesting biofluid for the search of biomarkers, as it is easily accessible in a noninvasive manner. However, given the incipient knowledge of salivary biomarkers in aging and its relationship to physical exercise, the present study is aimed at evaluating the protein expression and the levels of inflammatory and NETosis biomarkers in the saliva of practitioners (PE) and nonpractitioners (NPE) of physical exercise older adults. Six (6) practitioner and 4 nonpractitioner older adults were enrolled in this study. Unstimulated whole saliva was collected for analysis of the proteome by label-free mass spectrometry, as well as of the inflammatory status by evaluation of C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and cytokines (TNF-α, interleukin- (IL-) 1ß, and IL-8), while NETosis was assessed by myeloperoxidase (MPO) and neutrophil elastase. Regarding oral health, the decayed, missing, and filled teeth (DMF-T) index, bleeding on probing, suppuration, and probing depth measurement (mm) were evaluated. In addition, functional capacity was investigated using the General Physical Fitness Index (GPFI). In relation to the proteome analysis, 93 and 143 proteins were found exclusively in the PE and NPE groups, respectively; 224 proteins were common to both groups. Among these proteins, 10 proteins showed statistical difference (p < 0.05) between the groups: alpha-2-macroglobulin, component 3 of the complement, serotransferrin, and protein soluble in brain acid 1 were less expressed, while lactotransferrin, alpha-amylase 1, S100-A8, S100-A9, lactoperoxidase, and galectin-3 binding protein were more expressed in the PE group. No differences between groups were observed in the analysis of inflammatory and NETosis biomarkers. This study shows the potential utility of saliva for detecting protein biomarkers in a noninvasive biological sample of the elderly population.


Assuntos
Proteoma , Fator A de Crescimento do Endotélio Vascular , Idoso , Biomarcadores/metabolismo , Proteína C-Reativa/metabolismo , Exercício Físico , Humanos
4.
Cell Rep ; 39(5): 110764, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35508127

RESUMO

Linear motifs have an integral role in dynamic cell functions, including cell signaling. However, due to their small size, low complexity, and frequent mutations, identifying novel functional motifs poses a challenge. Viruses rely extensively on the molecular mimicry of cellular linear motifs. In this study, we apply systematic motif prediction combined with functional filters to identify human linear motifs convergently evolved also in viral proteins. We observe an increase in the sensitivity of motif prediction and improved enrichment in known instances. We identify >7,300 non-redundant motif instances at various confidence levels, 99 of which are supported by all functional and structural filters. Overall, we provide a pipeline to improve the identification of functional linear motifs from interactomics datasets and a comprehensive catalog of putative human motifs that can contribute to our understanding of the human domain-linear motif code and the associated mechanisms of viral interference.


Assuntos
Evolução Molecular , Proteoma , Motivos de Aminoácidos , Humanos , Proteoma/genética , Proteínas Virais/metabolismo
5.
Life Sci Alliance ; 5(8)2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35508387

RESUMO

The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase-substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity. Thirty-seven ALK substrate candidates, including 34 phosphorylation sites not annotated in the PhosphoSitePlus database, were identified by integrated analysis of the phosphoproteome and crosslinking interactome of HEK 293 cells with doxycycline-induced ALK overexpression. Furthermore, KSRs of ALK were validated by an in vitro kinase assay. Finally, using phosphoproteomic data from ALK mutant cell lines and patient-derived cells treated with ALK inhibitors, we found that the prediction of ALK activity was improved when the KSRs identified in this study were used instead of the public KSR dataset. Our approach is applicable to other kinases, and future identification of KSRs will facilitate more accurate estimations of kinase activity and elucidation of phosphorylation signals.


Assuntos
Proteoma , Transdução de Sinais , Quinase do Linfoma Anaplásico/metabolismo , Células HEK293 , Humanos , Fosforilação , Proteoma/metabolismo
6.
Methods Mol Biol ; 2469: 79-87, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35508831

RESUMO

Nuclei enrichment procedures enable a large variety of investigations. These studies include structural characterization of nuclear proteins, identification of posttranslational modifications, and regulation of stress or development-related gene expression. Successful enrichment of nuclei samples from plant tissues is crucial for a comprehensive analysis of the plant nuclear proteome. Here, we describe a method for nuclei enrichment from sugarcane stems and its assessment by western blot.


Assuntos
Saccharum , Núcleo Celular/metabolismo , Grão Comestível/química , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Saccharum/genética
7.
J Vis Exp ; (182)2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35499346

RESUMO

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.


Assuntos
Aminoacil-tRNA Sintetases , Proteoma , Aminoacil-tRNA Sintetases/genética , Animais , Cromatografia de Afinidade , Metionina , Camundongos , Proteostase
9.
Nat Commun ; 13(1): 2458, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513387

RESUMO

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce "Species by Proteome INvestigation" (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.


Assuntos
Proteoma , Proteômica , Animais , Arqueologia/métodos , Cromatografia Líquida , Mamíferos , Peptídeos , Proteômica/métodos , Espectrometria de Massas em Tandem
10.
Sci Rep ; 12(1): 7349, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513443

RESUMO

Recent spectacular advances by AI programs in 3D structure predictions from protein sequences have revolutionized the field in terms of accuracy and speed. The resulting "folding frenzy" has already produced predicted protein structure databases for the entire human and other organisms' proteomes. However, rapidly ascertaining a predicted structure's reliability based on measured properties in solution should be considered. Shape-sensitive hydrodynamic parameters such as the diffusion and sedimentation coefficients ([Formula: see text], [Formula: see text]) and the intrinsic viscosity ([η]) can provide a rapid assessment of the overall structure likeliness, and SAXS would yield the structure-related pair-wise distance distribution function p(r) vs. r. Using the extensively validated UltraScan SOlution MOdeler (US-SOMO) suite, a database was implemented calculating from AlphaFold structures the corresponding [Formula: see text], [Formula: see text], [η], p(r) vs. r, and other parameters. Circular dichroism spectra were computed using the SESCA program. Some of AlphaFold's drawbacks were mitigated, such as generating whenever possible a protein's mature form. Others, like the AlphaFold direct applicability to single-chain structures only, the absence of prosthetic groups, or flexibility issues, are discussed. Overall, this implementation of the US-SOMO-AF database should already aid in rapidly evaluating the consistency in solution of a relevant portion of AlphaFold predicted protein structures.


Assuntos
Proteoma , Bases de Dados de Proteínas , Humanos , Reprodutibilidade dos Testes , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
Sci Rep ; 12(1): 7314, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513538

RESUMO

Plasmalemmal ATP sensitive potassium (KATP) channels are recognized metabolic sensors, yet their cellular reach is less well understood. Here, transgenic Kir6.2 null hearts devoid of the KATP channel pore underwent multiomics surveillance and systems interrogation versus wildtype counterparts. Despite maintained organ performance, the knockout proteome deviated beyond a discrete loss of constitutive KATP channel subunits. Multidimensional nano-flow liquid chromatography tandem mass spectrometry resolved 111 differentially expressed proteins and their expanded network neighborhood, dominated by metabolic process engagement. Independent multimodal chemometric gas and liquid chromatography mass spectrometry unveiled differential expression of over one quarter of measured metabolites discriminating the Kir6.2 deficient heart metabolome. Supervised class analogy ranking and unsupervised enrichment analysis prioritized nicotinamide adenine dinucleotide (NAD+), affirmed by extensive overrepresentation of NAD+ associated circuitry. The remodeled metabolome and proteome revealed functional convergence and an integrated signature of disease susceptibility. Deciphered cardiac patterns were traceable in the corresponding plasma metabolome, with tissue concordant plasma changes offering surrogate metabolite markers of myocardial latent vulnerability. Thus, Kir6.2 deficit precipitates multiome reorganization, mapping a comprehensive atlas of the KATP channel dependent landscape.


Assuntos
NAD , Proteoma , Trifosfato de Adenosina , Coração , Canais KATP/genética , Canais KATP/metabolismo , NAD/metabolismo , Proteoma/metabolismo
12.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532271

RESUMO

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.


Assuntos
Proteômica , Análise de Célula Única , Animais , Linhagem da Célula , Espectrometria de Massas , Proteoma/metabolismo , Proteômica/métodos , Análise de Célula Única/métodos , Xenopus laevis/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(20): e2123411119, 2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35533274

RESUMO

SignificanceDietary fibers contain complex mixtures of biomolecules, making it difficult to develop/test hypotheses about how different fiber-types impact different components of the human gut microbiome and how microbiome changes that they produce are linked to human physiology. Here, we analyze microbiome and plasma proteome responses to consumption of two fiber-enriched snacks in two human studies. We use a variety of computational methods to correlate their effects on gut microbiome genes encoding enzymes that degrade complex fiber-associated polysaccharides, the microbial products of polysaccharide degradation, and plasma proteins representing diverse physiological processes. This approach can be used to guide the design of fiber-containing snacks that more precisely manipulate microbiome features in ways that improve nutritional and health status.


Assuntos
Microbioma Gastrointestinal , Microbiota , Fibras na Dieta/metabolismo , Microbioma Gastrointestinal/fisiologia , Humanos , Polissacarídeos/metabolismo , Proteoma
14.
Adv Protein Chem Struct Biol ; 130: 289-324, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35534110

RESUMO

Being phosphopeptide-binding hubs, 14-3-3 proteins coordinate multiple cellular processes in eukaryotes, including the regulation of apoptosis, cell cycle, ion channels trafficking, transcription, signal transduction, and hormone biosynthesis. Forming constitutive α-helical dimers, 14-3-3 proteins predominantly recognize specifically phosphorylated Ser/Thr sites within their partners; this generally stabilizes phosphotarget conformation and affects its activity, intracellular distribution, dephosphorylation, degradation and interactions with other proteins. Not surprisingly, 14-3-3 complexes are involved in the development of a range of diseases and are considered promising drug targets. The wide interactome of 14-3-3 proteins encompasses hundreds of different phosphoproteins, for many of which the interaction is well-documented in vitro and in vivo but lack the structural data that would help better understand underlying regulatory mechanisms and develop new drugs. Despite obtaining structural information on 14-3-3 complexes is still lagging behind the research of 14-3-3 interactions on a proteome-wide scale, recent works provided some advances, including methodological improvements and accumulation of new interesting structural data, that are discussed in this review.


Assuntos
Proteínas 14-3-3 , Fosfoproteínas , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Fosfoproteínas/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteoma/metabolismo
15.
Sci Rep ; 12(1): 7569, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534617

RESUMO

The tegument, as the surface layer of adult male and female Schistosoma spp. represents the protective barrier of the worms to the hostile environment of the host bloodstream. Here we present the first comparative analysis of sex-specific tegument proteins of paired or virgin Schistosoma mansoni. We applied a new and highly sensitive workflow, allowing detection of even low abundance proteins. Therefore, a streptavidin-biotin affinity purification technique in combination with single pot solid-phase enhanced sample preparation was established for subsequent LC-MS/MS analysis. We were able to identify 1519 tegument proteins for male and female virgin and paired worms and categorized them by sex. Bioinformatic analysis revealed an involvement of female-specific tegument proteins in signaling pathways of cellular processes and antioxidant mechanisms. Male-specific proteins were found to be enriched in processes linked to phosphorylation and signal transduction. This suggests a task sharing between the sexes that might be necessary for survival in the host. Our datasets provide a basis for further studies to understand and ultimately decipher the strategies of the two worm sexes to evade the immune system.


Assuntos
Proteoma , Schistosoma mansoni , Animais , Cromatografia Líquida , Feminino , Proteínas de Helminto/metabolismo , Masculino , Proteoma/metabolismo , Schistosoma mansoni/metabolismo , Espectrometria de Massas em Tandem
16.
Oxid Med Cell Longev ; 2022: 2590198, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35535361

RESUMO

The tryptophan residue has a large hydrophobic surface that plays a unique role in the folded protein conformation and functions. Tryptophan modifications are presumably to be readily detected in proteins due to the vulnerability of the indole structure to electrophilic attacks. In this study, we report a systematic identification of sequence variations at tryptophan, termed tryptophan variants, from the proteome of patients with nonsmall cell lung cancer (NSCLC). Using shotgun proteomics and a modified open search algorithm, 25 tryptophan variants on 2481 sites in over 858 proteins were identified. Among these, 6 tryptophan variants are previously identified, 15 are newly annotated, and 4 are still unknown, most of which are involved in the cascade of oxidation in the blood microparticle. Remarkably, Trp313 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-oxidized whereas Trp16 and Trp38 of hemoglobin (HBB) were down-oxidized in NCSLC tissues. The results were further supported by an independent cohort of 103 lung adenocarcinoma samples, reflecting a negative feedback and potential detoxification mechanism against tumor glycolysis and hypoxia. Overall, the study reports a quick approach to explore tryptophan variants at the proteomic scale. Our findings highlight the predominant role of tryptophan oxidation in regulating the redox balance of cancer cells and its potential role as prognostic biomarker for patients with NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Hipóxia , Neoplasias Pulmonares/patologia , Proteoma/metabolismo , Proteômica/métodos , Triptofano
17.
Sci Rep ; 12(1): 7497, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35523825

RESUMO

Alignment-free methods for sequence comparison and phylogeny inference have attracted a great deal of attention in recent years. Several algorithms have been implemented in diverse software packages. Despite the great number of existing methods, most of them are based on word statistics. Although they propose different filtering and weighting strategies and explore different metrics, their performance may be limited by the phylogenetic signal preserved in these words. Herein, we present a different approach based on the species-specific amino acid neighborhood preferences. These differential preferences can be assessed in the context of vector spaces. In this way, a distance-based method to build phylogenies has been developed and implemented into an easy-to-use R package. Tests run on real-world datasets show that this method can reconstruct phylogenetic relationships with high accuracy, and often outperforms other alignment-free approaches. Furthermore, we present evidence that the new method can perform reliably on datasets formed by non-orthologous protein sequences, that is, the method not only does not require the identification of orthologous proteins, but also does not require their presence in the analyzed dataset. These results suggest that the neighborhood preference of amino acids conveys a phylogenetic signal that may be of great utility in phylogenomics.


Assuntos
Aminoácidos , Proteoma , Algoritmos , Aminoácidos/genética , Filogenia , Alinhamento de Sequência , Software
18.
Sci Rep ; 12(1): 7421, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35523906

RESUMO

The molecular weight and isoelectric point of the proteins are very important parameters that control their subcellular localization and subsequent function. Although the genome sequence data of the plant kingdom improved enormously, the proteomic details have been poorly elaborated. Therefore, we have calculated the molecular weight and isoelectric point of the plant proteins and reported them in this database. A database, PlantMWpIDB, containing protein data from 342 plant proteomes was created to provide information on plant proteomes for hypothesis formulation in basic research and for biotechnological applications. The Molecular weight and isoelectric point (pI) are important molecular parameters of proteins that are useful when conducting protein studies involving 2D gel electrophoresis, liquid chromatography-mass spectrometry, and X-ray protein crystallography. PlantMWpIDB provides an easy-to-use and efficient interface for search options and generates a summary of basic protein parameters. The database represents a virtual 2D proteome map of plants, and the molecular weight and pI of a protein can be obtained by searching on the name of a protein, a keyword, or by a list of accession numbers. The PlantMWpIDB database also allows one to query protein sequences. The database can be found in the following link https://plantmwpidb.com/ . The individual 2D virtual proteome map of the plant kingdom will enable us to understand the proteome diversity between different species. Further, the molecular weight and isoelectric point of individual proteins can enable us to understand their functional significance in different species.


Assuntos
Proteoma , Proteômica , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Focalização Isoelétrica , Ponto Isoelétrico , Peso Molecular , Plantas/metabolismo , Proteoma/metabolismo , Proteômica/métodos
19.
Methods Mol Biol ; 2477: 261-274, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524122

RESUMO

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell. The use of semipermeabilized cells has several advantages, in particular the avoidance of generating fragile spheroplasts and the opportunity of employing cells from a glucose-containing medium for APEX2 tagging. In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions to achieve high-resolution mapping of a subcellular yeast proteome.


Assuntos
Proteômica , Saccharomyces cerevisiae , Ascorbato Peroxidases/química , Catálise , Marcação por Isótopo/métodos , Proteoma/metabolismo , Proteômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
Methods Mol Biol ; 2477: 275-292, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524123

RESUMO

Simple light isotope metabolic labeling (bSLIM) is an innovative method to accurately quantify differences in protein abundance at the proteome level in standard bottom-up experiments. The quantification process requires computation of the ratio of intensity of several isotopologs in the isotopic cluster of every identified peptide. Thus, appropriate bioinformatic workflows are required to extract the signals from the instrument files and calculate the required ratio to infer peptide/protein abundance. In a previous study (Sénécaut et al., J Proteome Res 20:1476-1487, 2021), we developed original open-source workflows based on OpenMS nodes implemented in a KNIME working environment. Here, we extend the use of the bSLIM labeling strategy in quantitative proteomics by presenting an alternative procedure to extract isotopolog intensities and process them by taking advantage of new functionalities integrated into the Minora node of Proteome Discoverer 2.4 software. We also present a graphical strategy to evaluate the statistical robustness of protein quantification scores and calculate the associated false discovery rates (FDR). We validated these approaches in a case study in which we compared the differences between the proteomes of two closely related yeast strains.


Assuntos
Proteoma , Proteômica , Marcação por Isótopo/métodos , Peptídeos/metabolismo , Proteômica/métodos , Saccharomyces cerevisiae/metabolismo
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