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1.
Sci Rep ; 11(1): 6621, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758289

RESUMEN

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.


Asunto(s)
Células Epiteliales/metabolismo , Proteoma/análisis , Proteómica/métodos , /metabolismo , Bronquios/citología , /virología , Técnicas de Cultivo de Célula , Línea Celular , Medios de Cultivo/química , Células Epiteliales/citología , Humanos , Espectrometría de Masas , Modelos Biológicos , Análisis de Componente Principal , /fisiología
2.
Ecotoxicol Environ Saf ; 214: 112083, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33676054

RESUMEN

Boron (B), an essential element for increasing seed yield and germinability in alfalfa (Medicago sativa L.), plays a vital role in its reproductive processes. However, effects of B stress on physiological and proteomic changes in reproductive organs related to alfalfa seed yield and germinability are poorly understood. In order to gain a better insight into B response or tolerance mechanisms, field trials were designed for B deficiency (0 mg B L-1), B sufficiency (800 mg B L-1), and B surplus (1600 mg B L-1) application during alfalfa flowering to analyze the proteomics and physiological responses of alfalfa 'Aohan' reproductive organs. Results showed that B deficiency weakened the stress-responsive ability in these organs, while B surplus reduced the sugar utilization of 'Aohan' flowers and caused lipid membrane peroxidation in 'Aohan' seeds. In addition, four upregulated stress responsive proteins (ADF-like protein, IMFP, NAD(P)-binding Rossmann-fold protein and NAD-dependent ALDHs) might play pivotal roles in the response of 'Aohan' reproductive organs to conditions of B deficiency and B surplus. All of the above results would be helpful to understand the tolerance mechanisms of alfalfa reproductive organs to both B deficiency and B surplus conditions, and also to give insight into the regulatory role of B in improving seed yield and germinability in alfalfa seed production. In summary, B likely plays a structural and regulatory role in relation to lipid metabolism, carbohydrate metabolism, amino acid metabolism, and signal transduction, thus regulates alfalfa reproductive processes eventually affecting the seed yield and germinability of alfalfa seeds.


Asunto(s)
Boro/fisiología , Medicago sativa/fisiología , Boro/metabolismo , Metabolismo de los Hidratos de Carbono , Flores , Genitales , Germinación , Medicago sativa/metabolismo , Proteómica/métodos , Semillas/metabolismo
3.
Methods Mol Biol ; 2259: 3-12, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687705

RESUMEN

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.


Asunto(s)
Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestructura , Células Madre Mesenquimatosas/citología , Proteínas/análisis , Células Cultivadas , Cromatografía Liquida/métodos , Medios de Cultivo/química , Humanos , Células Madre Mesenquimatosas/química , Microscopía Electrónica de Transmisión/métodos , Cultivo Primario de Células/métodos , Proteínas/aislamiento & purificación , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Cordón Umbilical/citología
4.
Methods Mol Biol ; 2259: 13-23, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687706

RESUMEN

In recent years, technical improvements in proteomics have allowed its rapid application for biomarker discovery, new drug target identification, and the study of disease progression and drug resistance. The clinical potential of circulating extracellular vesicles (EVs) as a source of biomarkers is one of the reasons why several research groups have recently applied proteomics to their study. A large variety of proteomic approaches such as gel-based proteomics and bottom-up and top-down mass spectrometry have been applied to the study of EVs. In this chapter, we will present basic protocols for gel-based and quantitative MS-based approaches applied to the study of EVs.


Asunto(s)
Proteínas Sanguíneas/análisis , Vesículas Extracelulares/química , Proteínas/análisis , Biomarcadores/análisis , Biomarcadores/sangre , Recolección de Muestras de Sangre/métodos , Humanos , Espectrometría de Masas/métodos , Proteómica/métodos , Electroforesis Bidimensional Diferencial en Gel/métodos
5.
Methods Mol Biol ; 2259: 25-45, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687707

RESUMEN

Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions-such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.-are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.


Asunto(s)
Extensiones de la Superficie Celular/química , Captura por Microdisección con Láser/métodos , Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Línea Celular , Fijadores , Humanos
6.
Methods Mol Biol ; 2259: 49-57, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687708

RESUMEN

Proteomics is one of the key approaches to understand plant cell physiology involving the regulation of expression of many genes and metabolite production. Technical advances allowed a deeper characterization of plant proteomes, highlighting the need to study cellular compartments. The apoplast is the cellular compartment external to the plasma membrane including the cell wall, where a broad range of processes take place including intercellular signaling, metabolite transport, and plant-microbe interactions. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves while maintaining cell integrity, which is particularly true for woody plants. Here, we describe the vacuum infiltration-centrifugation (VIC) method for the extraction of the apoplastic fluid compatible with high-throughput proteomic approaches and biochemical analysis from different woody plants.


Asunto(s)
Coffea/química , Hojas de la Planta/química , Proteínas de Plantas/aislamiento & purificación , Vitis/química , Pared Celular/química , Centrifugación/métodos , Proteínas de Plantas/análisis , Proteómica/métodos , Vacio
7.
Methods Mol Biol ; 2259: 59-75, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687709

RESUMEN

Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are present in many fish species and cephalopods all over the globe, might be consumed and develop occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and economic concern. In recent years, proteomic methods have gained greater acceptance among scientists involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)-based shotgun proteomics to define differences in proteomic composition between L3 and L4 development stages of A. simplex s.s.


Asunto(s)
Anisakis/crecimiento & desarrollo , Proteínas del Helminto/análisis , Proteómica/métodos , Animales , Anisakiasis/parasitología , Anisakis/química , Anisakis/metabolismo , Cromatografía Liquida/métodos , Proteínas del Helminto/metabolismo , Humanos , Larva/química , Larva/crecimiento & desarrollo , Larva/metabolismo , Espectrometría de Masas en Tándem/métodos
8.
Methods Mol Biol ; 2259: 77-102, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687710

RESUMEN

During the last decade, we have witnessed outstanding advances in proteomics led mostly by great technological improvements in mass spectrometry field allowing high-throughput production of high-quality data used for massive protein identification and quantification. From a practical viewpoint, these advances have been mainly exploited in research projects involving model organisms with abundant genomic and proteomic information available in public databases. However, there is a growing number of organisms of high interest in different disciplines, such as ecological, biotechnological, and evolutionary research, yet poorly represented in these databases. Important advances in massive parallel sequencing technology and easy accessibility of this technology to many research laboratories have made nowadays possible to produce customized genomic and proteomic databases of any organism. Along this line, the use of proteogenomic approaches by combining in the same analysis the data obtained from different omic levels has emerged as a very useful and powerful strategy to run shotgun proteomic experiments specially focused on non-model organisms. In this chapter, we provide detailed procedures to undertake shotgun quantitative proteomic experiments following either a label-free or an isobaric labeling approach in non-model organisms, emphasizing also a few key aspects related to experimental design and data analysis.


Asunto(s)
Proteoma/análisis , Proteómica/métodos , Animales , Fraccionamiento Químico/métodos , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Proteoma/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos
9.
Methods Mol Biol ; 2259: 143-151, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687712

RESUMEN

The versatility of protein microarrays provides researchers with a wide variety of possibilities to address proteomic studies. Therefore, protein microarrays are becoming very useful tools to identify candidate biomarkers in human body fluids for disease states such as rheumatoid arthritis (RA). In RA serum, there is a high prevalence of rheumatoid factor (RF), which is an antibody with high specificity against Fc portion of IgG. The presence of RF, in particular RF-IgM, has the great potential to interfere with antibody-based immunoassays by nonspecifically binding capture antibodies. Because of this concern, we describe a procedure to reduce the interference of RF-IgM on RA serum protein profiling approaches based on multiplexed antibody suspension bead arrays.


Asunto(s)
Artritis Reumatoide/sangre , Proteómica/métodos , Anticuerpos Inmovilizados/química , Artritis Reumatoide/diagnóstico , Biomarcadores/análisis , Biomarcadores/sangre , Humanos , Inmunoglobulina M/análisis , Inmunoglobulina M/sangre , Factor Reumatoide/análisis , Factor Reumatoide/sangre
10.
Methods Mol Biol ; 2259: 105-141, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687711

RESUMEN

Identification of molecular biomarkers for human diseases is one of the most important disciplines in translational science as it helps to elucidate their origin and early progression. Thus, it is a key factor in better diagnosis, prognosis, and treatment. Proteomics can help to solve the problem of sample complexity when the most common primary sample specimens were analyzed: organic fluids of easy access. The latest developments in high-throughput and label-free quantitative proteomics (SWATH-MS), together with more advanced liquid chromatography, have enabled the analysis of large sample sets with the sensitivity and depth needed to succeed in this task. In this chapter, we show different sample processing methods (major protein depletion, digestion, etc.) and a micro LC-SWATH-MS protocol to identify/quantify several proteins in different types of samples (serum/plasma, saliva, urine, tears).


Asunto(s)
Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/orina , Proteínas Sanguíneas/análisis , Humanos , Espectrometría de Masas/métodos , Proteinuria/diagnóstico , Saliva/química , Manejo de Especímenes/métodos , Lágrimas/química
11.
Methods Mol Biol ; 2259: 153-165, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687713

RESUMEN

Proteomic tools are especially useful when it comes to investigating complex samples such as human blood plasma, in which protein quantities can span across up to ten orders of magnitude. Ultra definition mass spectrometry, in combination with two-dimensional liquid chromatography, provides better coverage of complex proteomes and allows for better control of collision energy, keeping the fragmentation benefits of high collision energy associated with drift time measurements from ion mobility separation. Here, we present a protocol to assist in the identification of proteins in human blood plasma and other similar samples with a large dynamic range.


Asunto(s)
Proteínas Sanguíneas/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Cromatografía de Afinidad/métodos , Humanos , Programas Informáticos
12.
Methods Mol Biol ; 2259: 167-179, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687714

RESUMEN

Metaproteomics of host-microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification.Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography-mass spectrometry, and data analysis.


Asunto(s)
Bacterias/aislamiento & purificación , Proteínas Bacterianas/análisis , Proteínas Fúngicas/análisis , Hongos/aislamiento & purificación , Microbioma Gastrointestinal , Proteómica/métodos , Animales , Cromatografía Liquida/métodos , Intestinos/microbiología , Espectrometría de Masas/métodos , Ratones , Péptidos/análisis , Flujo de Trabajo
13.
Methods Mol Biol ; 2259: 181-189, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687715

RESUMEN

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."


Asunto(s)
Proteínas Bacterianas/análisis , Mycobacterium/química , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Mycobacterium/citología , Infecciones por Mycobacterium/microbiología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/citología , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos
14.
Methods Mol Biol ; 2259: 205-213, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687717

RESUMEN

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.


Asunto(s)
Bacterias/aislamiento & purificación , Proteínas Bacterianas/análisis , Microbiología de Alimentos , Proteómica/métodos , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida/métodos , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos
15.
Methods Mol Biol ; 2259: 215-223, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687718

RESUMEN

A workflow for the characterization of food-derived bioactive peptides is described in this chapter. The workflow integrates two consecutive steps: a discovery phase and a protein-based bioinformatic phase. In the first step (discovery phase), a shotgun bottom-up proteomics approach is used to create a reference data set for a selected food proteome. Afterward, in a second step (bioinformatic phase), the reference proteome is subjected to several in silico protein-based bioinformatic analyses to predict and characterize potential bioactive peptides after an in silico human gastrointestinal digestion. Using this workflow, bioactive collagen peptides, antihypertensive, antimicrobial, and antitumor peptides were predicted as potential valuable bioactive peptides from seafood and marine by-products. It is concluded that the combination of the global shotgun proteomic analysis and the analysis by protein-based bioinformatics can provide a rapid strategy for the characterization of new potential food-derived bioactive peptides.


Asunto(s)
Proteínas en la Dieta/análisis , Péptidos/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Alimentos Funcionales/análisis , Humanos , Alimentos Marinos/análisis , Espectrometría de Masas en Tándem/métodos
16.
Methods Mol Biol ; 2259: 227-246, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687719

RESUMEN

Carbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC-MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability.


Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Carbonilación Proteica , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Focalización Isoeléctrica/métodos , Oxidación-Reducción , Proteoma/análisis
17.
Methods Mol Biol ; 2259: 247-257, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687720

RESUMEN

Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.


Asunto(s)
Fosfopéptidos/análisis , Proteómica/métodos , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoprecipitación/métodos , Células MCF-7 , Fosfopéptidos/aislamiento & purificación , Proteoma/análisis , Proteoma/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos
18.
Methods Mol Biol ; 2259: 259-268, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687721

RESUMEN

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.


Asunto(s)
Fosfopéptidos/análisis , Fosfoproteínas/análisis , Proteómica/métodos , Fraccionamiento Químico/métodos , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Humanos , Células Jurkat , Fosfopéptidos/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosforilación , Proteoma/análisis , Proteoma/aislamiento & purificación , Proteómica/economía , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/métodos , Factores de Tiempo , Titanio/química , Flujo de Trabajo
19.
Methods Mol Biol ; 2259: 269-294, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687722

RESUMEN

In living cells, most proteins are organized in stable or transient functional assemblies, protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. Over several decades, specific protein complexes have been analyzed by structural biology methods, initially X-ray crystallography and more recently single particle cryoEM. In parallel, mass spectrometry (MS)-based methods including in vitro affinity-purification coupled to MS or in vivo protein proximity-dependent labeling methods have proven particularly effective to detect complexes, thus nominating new assemblies for structural analysis. Those approaches, however, are either of limited in throughput or require specifically engineered protein systems.In this chapter, we present protocols for a workflow that supports the parallel analysis of multiple complexes from the same biological sample with respect to abundance, subunit composition, and stoichiometry. It consists of the separation of native complexes by size-exclusion chromatography (SEC) and the subsequent mass spectrometric analysis of the proteins in consecutive SEC fractions. In particular, we describe (1) optimized conditions to achieve native protein complex separation by SEC, (2) the preparation of the SEC fractions for MS analysis, (3) the acquisition of the MS data at high throughput via SWATH/DIA (data-independent analysis) mass spectrometry and short chromatographic gradients, and (4) a set of bioinformatic tools for the targeted analysis of protein complexes. Altogether, the parallel measurement of a high number of complexes from a single biological sample results in unprecedented system-level insights into the remodeling of cellular protein complexes in response to perturbations of a broad range of cellular systems.


Asunto(s)
Cromatografía en Gel/métodos , Espectrometría de Masas/métodos , Proteínas/análisis , Proteómica/métodos , Cromatografía Líquida de Alta Presión/métodos , Humanos , Células Jurkat , Ultracentrifugación/métodos , Flujo de Trabajo
20.
Methods Mol Biol ; 2259: 297-308, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687723

RESUMEN

Shotgun proteomics is the inferential analysis of proteoforms using peptide proxies produced by enzyme-catalyzed hydrolysis of entire proteomes. Such peptides are usually identified by nanoflow liquid chromatography coupled to tandem mass spectrometry analysis (nLC-MS/MS). Traditionally, MS/MS analysis is performed in data-dependent acquisition (DDA) mode, which usually produces a pattern of fragment masses unique to a single peptide's fragmentation. Here, I describe a statistically rigorous qualitative and quantitative computational analysis for shotgun proteomics DDA analysis using free open-source software tools. MS/MS data are used to identify peptides, and the area of peptide mass/charge over chromatographic elution is used to quantify peptides. All peptides that uniquely map to a protein sequence predicted from the genome are combined into a single protein quantity, which can then be compared across experimental conditions. Statistically significant protein changes can be summarized using gene ontology or pathway term enrichment analysis.


Asunto(s)
Fragmentos de Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Ontología de Genes , Humanos , Anotación de Secuencia Molecular , Proteoma/análisis , Programas Informáticos
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