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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.
Analyst ; 146(21): 6621-6630, 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34591044

RESUMO

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.


Assuntos
Microfluídica , Proteínas , Eletroforese , Hidrogéis , Imunoensaio
3.
J Am Chem Soc ; 142(42): 17938-17943, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33022172

RESUMO

A nanoparticle can hold multiple types of therapeutic and imaging agents for disease treatment and diagnosis. However, controlling the storage of molecules in nanoparticles is challenging, because nonspecific intermolecular interactions are used for encapsulation. Here, we used specific DNA interactions to store molecules in nanoparticles. We made nanoparticles containing DNA anchors to capture DNA-conjugated small molecules. By changing the sequences and stoichiometry of DNA anchors, we can control the amount and ratio of molecules with different chemical properties in the nanoparticles. We modified the cytotoxicity of our nanoparticles to cancer cells by changing the ratio of encapsulated drugs (mertansine and doxorubicin). Specifically controlling the storage of multiple types of molecules allows us to optimize the properties of combination drug and imaging nanoparticles.


Assuntos
DNA/química , Nanopartículas/química , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Células HeLa , Humanos , Maitansina/farmacologia , Imagem Óptica , Tamanho da Partícula , Propriedades de Superfície
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 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
6.
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
7.
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
8.
Proc Natl Acad Sci U S A ; 102(52): 19208-13, 2005 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-16365317

RESUMO

Given the mounting evidence for involvement of delta opioid receptors in the tolerance and physical dependence of mu opioid receptor agonists, we have investigated the possible physical interaction between mu and delta opioid receptors by using bivalent ligands. Based on reports of suppression of antinociceptive tolerance by the delta antagonist naltrindole (NTI), bivalent ligands [mu-delta agonist-antagonist (MDAN) series] that contain different length spacers, and pharmacophores derived from NTI and the mu agonist oxymorphone, have been synthesized and evaluated by intracerebroventricular (i.c.v.) administration in the tail-flick test in mice. In acute i.c.v. studies, the bivalent ligands functioned as agonists with potencies ranging from 1.6- to 45-fold greater than morphine. In contrast, the monovalent mu agonist analogues were substantially more potent than the MDAN congeners and were essentially equipotent with one another and oxymorphone. Pretreatment with NTI decreased the ED(50) values for MDAN-19 to a greater degree than for MDAN-16 but had no effect on MDAN-21. Chronic i.c.v. studies revealed that MDAN ligands whose spacer was 16 atoms or longer produced less dependence than either morphine or mu monovalent control MA-19. On the other hand, both physical dependence and tolerance were suppressed at MDAN spacer lengths of 19 atoms or greater. These data suggest that physical interaction between the mu and delta opioid receptors modulates mu-mediated tolerance and dependence. Because MDAN-21 was found to be 50-fold more potent than morphine by the i.v. route (i.v.), it offers a previously uncharacterized approach for the development of analgesics devoid of tolerance and dependence.


Assuntos
Analgésicos/farmacologia , Naltrexona/análogos & derivados , Entorpecentes/metabolismo , Analgésicos Opioides/farmacologia , Animais , Química Farmacêutica/métodos , Relação Dose-Resposta a Droga , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Químicos , Modelos Estatísticos , Morfina/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides delta/química , Receptores Opioides mu/química , Fatores de Tempo
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