RESUMEN
Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.
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Péptidos/química , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Línea Celular , Humanos , Marcaje IsotópicoRESUMEN
Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.
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Proteínas de Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Arabidopsis/genética , Daño del ADN , Redes y Vías Metabólicas , Mutación , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteómica , Transducción de Señal , Estrés FisiológicoRESUMEN
The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.
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Proteoma , Proteómica , Línea Celular , Indicadores y Reactivos , PéptidosRESUMEN
Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+. We label each peptide with TMTproZero or Super Heavy TMTpro reagents and separately spike each peptide into a TMTpro16-labeled background (equal amount of peptide across all 16 channels). We observe that the expected 1:1 reporter ion ratio is distorted when a TMTproZero-labeled peptide is used; however, we note no such interference when shTMTpro substitutes the TMTproZero tag. Our data suggest that using the Super Heavy TMTpro reagent is an improvement over the TMTproZero reagent for the accurate quantification of high-charge-state peptides for trigger-based multiplexed assays.
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Proteómica , Espectrometría de Masas en Tándem , Indicadores y Reactivos , Marcaje Isotópico , IsótoposRESUMEN
RATIONALE: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS: We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. CONCLUSIONS: Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.
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Biotina/metabolismo , Cisteína/metabolismo , Sondas Moleculares , Miocardio/metabolismo , Compuestos Nitrosos/metabolismo , Proteómica/métodos , Alcohol Deshidrogenasa/deficiencia , Alcohol Deshidrogenasa/genética , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nitrosación , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.
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Glicómica/métodos , Oximas/química , Piperidinas/química , Polisacáridos/análisis , Biomarcadores/análisis , Línea Celular Tumoral , Cromatografía Liquida/métodos , Enfermedades del Esófago/sangre , Fetuínas/química , Glicoproteínas/química , Humanos , Ribonucleasas/química , Espectrometría de Masas en Tándem/métodosRESUMEN
Mass spectrometry (MS) has become the predominant technology to analyze proteins due to it ability to identify and characterize proteins and their modifications with high sensitivity and selectivity (Aebersold and Mann, Nature 422(6928):198-207, 2003; Han et al., Curr Opin Chem Biol 12(5):483-490, 2008). While mass spectrometry instruments have improved rapidly over the past couple of decades, mass spectrometry results have remained largely dependent on sample preparation and quality. Sample ionization and mass measurements are susceptible to a wide variety of interferences, including buffers, salts, polymers, and detergents. These contaminants also impair MS system performance, often requiring time consuming maintenance or costly repairs to restore function. The goal of this chapter is to describe the rationale, considerations, and general techniques used to prepare samples for proteomic mass spectrometry analysis.
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Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Proteoma , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Fraccionamiento Celular , Cromatografía Liquida , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , ProteolisisRESUMEN
Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.
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Electroforesis Capilar/métodos , Polisacáridos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography-tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.
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Yodoacetatos/química , Animales , Línea Celular , Lipopolisacáridos/farmacología , Ratones , Anotación de Secuencia Molecular , Mapeo Peptídico , Procesamiento Proteico-Postraduccional , Proteómica , Coloración y EtiquetadoRESUMEN
N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 µM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.
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Polisacáridos/análisis , Proteoma/química , Proteómica/métodos , Animales , Línea Celular Tumoral , Glicoproteínas/química , Humanos , Estructura Molecular , Polisacáridos/química , Carbonilación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a (15)N for a (13)C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a (13)C was exchanged for a (15)N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the (13)C- and (15)N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex.
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Cromatografía Líquida de Alta Presión , Proteoma/análisis , Espectrometría de Masas en Tándem , Animales , Encéfalo/metabolismo , Isótopos de Carbono/química , Células HeLa , Humanos , Ratones , Isótopos de Nitrógeno/química , Bazo/metabolismo , Tiazoles/químicaRESUMEN
Detergents are commonly used in protein-chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1-5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography-tandem MS (LC-MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)-MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15min), efficient detergent removal, and high recovery of proteins and peptides.
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Fraccionamiento Químico/instrumentación , Detergentes/aislamiento & purificación , Oligosacáridos/química , Péptidos/química , Proteínas/química , Resinas Sintéticas/química , Animales , Bovinos , Cromatografía Liquida , Células HeLa , Humanos , Espectrometría de Masas , Albúmina Sérica Bovina/química , Propiedades de Superficie , Tripsina/químicaRESUMEN
Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.
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Glioblastoma/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-6/metabolismo , Células Madre Neoplásicas/química , Fosfoproteínas/análisis , Proteoma/análisis , Factor de Transcripción STAT3/metabolismo , Western Blotting , Quimiocinas/metabolismo , Cromatografía Liquida/métodos , Glioblastoma/metabolismo , Humanos , Hipoxia/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteoma/metabolismo , Transducción de Señal , Espectrometría de Masas en Tándem/métodos , Triptófano/metabolismoRESUMEN
Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200 fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested.
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Proteínas Sanguíneas/química , Animales , Precipitación Química , Haplorrinos , Humanos , Factor Plaquetario 4/sangre , Sustancias Reductoras/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug-drug interactions with other medicines if developed as therapeutic agents.
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Amidohidrolasas/antagonistas & inhibidores , Benzamidas/farmacología , Carbamatos/farmacología , Carboxilesterasa/metabolismo , Inhibidores Enzimáticos/farmacología , Amidohidrolasas/química , Amidohidrolasas/inmunología , Animales , Anticuerpos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Corazón/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/enzimología , Hígado/efectos de los fármacos , Hígado/enzimología , Proteoma/efectos de los fármacos , Proteoma/metabolismo , Ratas , Transfección/métodosRESUMEN
Cancer forms specialized microenvironmental niches that promote local invasion and colonization. Engrafted patient-derived xenografts (PDXs) locally invade and colonize naïve stroma in mice while enabling unambiguous molecular discrimination of human proteins in the tumor from mouse proteins in the microenvironment. To characterize how patient breast tumors form a niche and educate naïve stroma, subcutaneous breast cancer PDXs were globally profiled by species-specific quantitative proteomics. Regulation of PDX stromal proteins by breast tumors was extensive, with 35% of the stromal proteome altered by tumors consistently across different animals and passages. Differentially regulated proteins in the stroma clustered into six signatures, which included both known and previously unappreciated contributors to tumor invasion and colonization. Stromal proteomes were coordinately regulated; however, the sets of proteins altered by each tumor were highly distinct. Integrated analysis of tumor and stromal proteins, a comparison made possible in these xenograft models, indicated that the known hallmarks of cancer contribute pleiotropically to establishing and maintaining the microenvironmental niche of the tumor. Education of the stroma by the tumor is therefore an intrinsic property of breast tumors that is highly individualized, yet proceeds by consistent, nonrandom, and defined tumor-promoting molecular alterations.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteoma/metabolismo , Microambiente Tumoral , Animales , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteoma/análisis , Proteoma/genética , Proteómica , Células del Estroma/metabolismo , Células del Estroma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein carbonylation is a common oxidative stress (OS)-driven post-translational modification (PTM). Proteome-wide carbonylation events can best be characterized using a combination of analytical approaches. Immunoblotting of carbonylated proteins provides data on the extent of modifications within complex samples, as well as a broad comparison of carbonylation profiles between different biological states (e.g., disease versus control), while mass spectrometry (MS)-based analysis provides information on proteins susceptible to carbonylation, as well as the potential for quantitative characterization of specific sites of amino acid modification. Here, we present a novel use for aminoxyTMT, a derivative of the Tandem Mass Tag (TMT) isobaric labeling reagent, which utilizes an aminooxy functional group for covalent labeling of reactive carbonyls in proteins. When coupled with anti-TMT antibody, we demonstrate the use of aminoxyTMT for immunoblot profiling of protein carbonylation in complex mixtures, as well as enrichment of modified peptides from these mixtures. Proof-of-principle experiments also show the amenability of aminoxyTMT-labeled carbonylated peptides enriched from complex mixtures to identification using tandem MS (MS/MS) and database searching, as well as quantitative analysis using TMT-based reporter ion intensity measurements.
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Immunoblotting/métodos , Espectrometría de Masas/métodos , Carbonilación Proteica , Proteínas/química , Secuencia de Aminoácidos , Animales , Humanos , Indicadores y Reactivos/química , Ratones , Péptidos/químicaRESUMEN
BACKGROUND AND OBJECTIVES: Reflection after patient encounters is an important aspect of clinical learning. After our medical school instituted a reflection paper assignment for all clerkships, we wanted to learn about the types of encounters that students found meaningful on a family medicine clerkship and how they impacted students' learning. METHODS: Family and Community Medicine Clerkship students completed a reflection paper after the clerkship, based on guidelines that were used for all clerkship reflection papers at our medical school. Two reviewers independently organized student responses into themes and then jointly prioritized common themes and negotiated any initial differences into other themes. RESULTS: A total of 272 reflection papers describing an actual learning moment in patient care were submitted during the study period of January 2011--December 2012. In describing actions performed, students most frequently wrote about aspects of patient-centered care such as listening to the patient, carefully assessing the patient's condition, or giving a detailed explanation to the patient. In describing effects of those actions, students wrote about what they learned about the patient-physician interaction, the trust that patients demonstrated in them, the approval they gained from their preceptors, and the benefits they saw from their actions. CONCLUSIONS: An important contribution of a family medicine clerkship is the opportunity for students to further their skills in patient-centered care and realize the outcomes of providing that type of care.
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Prácticas Clínicas , Medicina Familiar y Comunitaria/educación , Relaciones Médico-Paciente , Estudiantes de Medicina , Competencia Clínica , Humanos , Aprendizaje , Educación del Paciente como Asunto , Atención Dirigida al Paciente , Estudiantes de Medicina/psicología , Evaluación de Síntomas , ConfianzaRESUMEN
Protein glycosylation is a common post-translational modification, which serves critical roles in the biological processes of organisms. Monitoring of changes in the abundance and structure of glycans may be necessary to explain the correlations between protein glycosylation and various diseases. Hence, the growing importance of glycoproteomics necessitates in-depth qualitative and quantitative studies of glycans. One of the emerging trends in glycomics research is the innovation related to accurate mass spectrometry based quantitative analysis of glycans. Recently, we have introduced aminoxyTMT reagents, which enable efficient relative quantitation of carbohydrates, improved glycan ionization efficiency and increased analytical throughput. These reagents can be used for quantitative analysis of N-glycans by direct infusion or liquid chromatography (LC)-coupled to electrospray ionization mass spectrometry (ESI-MS). However, unlike in proteomics, one of the major challenges left unaddressed is the lack of informatics tools to automate the qualitative and quantitative analysis of generated data. This analysis typically includes identification/quantitation of glycans using MS/MS data and differential analysis across biological samples. We have developed software modules to streamline such protocols for quantitative analysis of aminoxyTMT labeled-glycans derived from complex mixtures. This article is part of a Special Issue entitled: Proteomics in India.