Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-34927332

RESUMO

Cross-linking mass spectrometry (XL-MS) is an attractive method for the proteome-wide characterization of protein structures and interactions. Currently, the depth of in vivo XL-MS studies is lagging behind the established applications to cell lysates, because cross-linking reagents that can penetrate intact cells and strategies to enrich cross-linked peptides lack in efficiency. To tackle these limitations, we develop a phosphonate-containing cross-linker, tBu-PhoX that efficiently permeates various biological membranes and can be robustly enriched using routine Immobilized Metal Ion Affinity Chromatography. We establish a tBu-PhoX-based in vivo XL-MS pipeline, achieving high cross-link identification numbers in intact human cells with substantially reduced analysis time. Collectively, the developed cross-linker and XL-MS pipeline pave the way for comprehensive XL-MS characterization of living systems.

2.
J Proteome Res ; 20(5): 2964-2972, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33900084

RESUMO

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.


Assuntos
Proteoma , Proteômica , Linhagem Celular , Indicadores e Reagentes , Peptídeos
3.
J Proteome Res ; 20(5): 3009-3013, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33689365

RESUMO

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.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Indicadores e Reagentes , Marcação por Isótopo , Isótopos
4.
Nat Methods ; 17(4): 399-404, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203386

RESUMO

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.


Assuntos
Peptídeos/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Humanos , Marcação por Isótopo
5.
J Proteome Res ; 18(2): 594-605, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30501201

RESUMO

Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) is a recently introduced targeted proteomics method that combines peptide and sample multiplexing. TOMAHAQ assays enable sensitive and accurate multiplexed quantification by implementing an intricate data collection scheme that comprises multiple MSn scans, mass inclusion lists, and data-driven filters. Consequently, manual creation of TOMAHAQ methods can be time-consuming and error prone, while the resulting TOMAHAQ data may not be compatible with common mass spectrometry analysis pipelines. To address these concerns we introduce TomahaqCompanion, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of TOMAHAQ data. Starting from a list of peptide sequences, a user can perform each step of TOMAHAQ assay development including (1) generation of priming run target list, (2) analysis of priming run data, (3) generation of TOMAHAQ method file, and (4) analysis and export of quantitative TOMAHAQ data. We demonstrate the flexibility of TomahaqCompanion by creating a variety of methods testing TOMAHAQ parameters (e.g., number of SPS notches, run length, etc.). Lastly, we analyze an interference sample comprising heavy yeast peptides, a standard human peptide mixture, TMT11-plex, and super heavy TMT (shTMT) isobaric labels to demonstrate ∼10-200 attomol limit of quantification within a complex background using TOMAHAQ.


Assuntos
Peptídeos/análise , Proteômica/métodos , Humanos , Software , Coloração e Rotulagem , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo , Interface Usuário-Computador , Leveduras
6.
J Proteome Res ; 16(11): 4244-4252, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29022350

RESUMO

Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs) has become a powerful platform for simultaneous, large-scale phospho-proteome site identification and quantitation. To improve the accuracy of isobaric tag-based quantitation in complex proteomic samples, MS3-based acquisition methods such as Synchronous Precursor Selection (SPS) have been used. However, the method suffers from lower peptide identification rates when applied to enriched phosphopeptide samples compared with unmodified samples due to differences in phosphopeptide fragmentation patterns during tandem MS. We developed and optimized two new acquisition methods for analysis of TMT-labeled multiplexed phosphoproteome samples, which resulted in more phosphopeptide identifications with less ratio distortion when compared with previous methods. We also applied these improved methods to a large-scale study of phosphorylation levels in A549 cell lines treated with insulin or insulin growth factor 1 (IGF-1). Overall, 3378 protein groups and 12 465 phosphopeptides were identified, of which 10 436 were quantified across 10 samples without prefractionation. The accurate measurement enabled us to map to numerous signaling pathways including mechanistic target of rapamycin (mTOR), epidermal growth factor receptor (EGFR, ErbB), and insulin signaling pathways.


Assuntos
Fosfopeptídeos/análise , Coloração e Rotulagem/métodos , Células A549 , Receptores ErbB/metabolismo , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Métodos , Fosforilação , Transdução de Sinais , Coloração e Rotulagem/normas , Serina-Treonina Quinases TOR/metabolismo
7.
J Am Soc Mass Spectrom ; 28(10): 2011-2021, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28799075

RESUMO

Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments. Graphical Abstract ᅟ.


Assuntos
Aminoácidos/química , Diazometano/química , Metano/análogos & derivados , Proteínas/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida , Indicadores e Reagentes/química , Isomerismo , Metano/química , Fotólise
8.
Sci Signal ; 10(491)2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28790197

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteoma/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Metástase Neoplásica , Proteoma/análise , Proteoma/genética , Proteômica , Células Estromais/metabolismo , Células Estromais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Adv Exp Med Biol ; 919: 43-62, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27975212

RESUMO

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.


Assuntos
Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Fracionamento Celular , Cromatografia Líquida , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Proteólise
10.
PLoS One ; 11(6): e0157762, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27336622

RESUMO

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC's utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC's efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/administração & dosagem , Imunoconjugados/uso terapêutico , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Linhagem Celular Tumoral , Corantes Fluorescentes/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Nus , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/diagnóstico por imagem , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Biotechniques ; 60(4): 186-8, 190, 192-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27071607

RESUMO

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.


Assuntos
Immunoblotting/métodos , Espectrometria de Massas/métodos , Carbonilação Proteica , Proteínas/química , Sequência de Aminoácidos , Animais , Humanos , Indicadores e Reagentes/química , Camundongos , Peptídeos/química
12.
Circ Res ; 117(10): 846-57, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26338901

RESUMO

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.


Assuntos
Biotina/metabolismo , Cisteína/metabolismo , Sondas Moleculares , Miocárdio/metabolismo , Compostos Nitrosos/metabolismo , Proteômica/métodos , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrosação , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
13.
J Proteome Res ; 13(7): 3200-11, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24926564

RESUMO

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.


Assuntos
Iodoacetatos/química , Animais , Linhagem Celular , Lipopolissacarídeos/farmacologia , Camundongos , Anotação de Sequência Molecular , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Proteômica , Coloração e Rotulagem
14.
Antioxid Redox Signal ; 20(9): 1365-81, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24152285

RESUMO

AIMS: Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities. RESULTS: We have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect. INNOVATION: We demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels. CONCLUSION: We have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection.


Assuntos
Cisteína/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Proteômica , Animais , Hipóxia Celular , Linhagem Celular , Dissulfeto de Glutationa/metabolismo , Espectrometria de Massas , Oxirredução , Proteômica/métodos , Ratos , S-Nitrosoglutationa/metabolismo
15.
Anal Chem ; 84(10): 4411-8, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22480364

RESUMO

Carbene chemistry has been used recently in structural mass spectrometry as a labeling method for mapping protein surfaces. The current study presents a method for quantitating label distribution at the amino acid level and explores the nature and basis for an earlier observation of labeling bias. With the use of a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) applied to digests of holo-calmodulin, we developed a quantitation strategy to map site-specific incorporation of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic acid and 4,4-azipentanoic acid. The approach provides reliable incorporation data for fragments generated by electron-transfer dissociation, whereas high-energy collisional dissociation leads to energy and sequence-dependent loss of the label as a neutral. However, both can produce data suitable for mapping residues in the interaction of holo-calmodulin with M13 peptide ligand. Site-specific labeling was monitored as a function of reagent, ionic strength, and temperature, demonstrating that electrostatic interactions at the protein surface can "steer" the distribution of label precursors to sites of surface charge and favor label insertion into residues in the vicinity of the surface charge. A further preference for insertion into carboxylates was observed, based on chemical reactivity. We suggest that decoupling surface partitioning from the chemistry of insertion offers a flexible, tunable labeling strategy for structural mass spectrometry that can be applied to a broad range of protein surface compositions and promotes the design of reagents to simplify the workflow.


Assuntos
Cromatografia Líquida de Alta Pressão , Metano/análogos & derivados , Peptídeos/análise , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Metano/química , Fotólise , Pegadas de Proteínas , Eletricidade Estática
16.
Anal Chem ; 84(8): 3716-24, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22455665

RESUMO

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.


Assuntos
Polissacarídeos/análise , Proteoma/química , Proteômica/métodos , Animais , Linhagem Celular Tumoral , Glicoproteínas/química , Humanos , Estrutura Molecular , Polissacarídeos/química , Carbonilação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
EMBO J ; 25(11): 2605-14, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16675950

RESUMO

An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/efeitos da radiação , Transdução de Sinais/fisiologia , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Camundongos , Oócitos/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Raios Ultravioleta , Proteínas de Xenopus , Xenopus laevis , Proteína de Xeroderma Pigmentoso Grupo A/genética
18.
J Biol Chem ; 279(14): 13346-53, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-14724280

RESUMO

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Celulose/genética , Cromatina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Exodesoxirribonucleases/química , Humanos , Técnicas In Vitro , Rim/citologia , Peso Molecular , Fosfoproteínas/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...