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1.
Sci Rep ; 14(1): 4985, 2024 02 29.
Article de Anglais | MEDLINE | ID: mdl-38424293

RÉSUMÉ

Cysteine protease inhibitor 1 (CST1) is a cystatin superfamily protein that inhibits cysteine protease activity and is reported to be involved in the development of many malignancies. Mitochondrial oxidative phosphorylation (OXPHOS) also plays an important role in cancer cell growth regulation. However, the relationship and roles of CST1 and OXPHOS in esophageal squamous cell carcinoma (ESCC) remains unclear. In our pilot study, CST1 was shown the potential of promoting ESCC migration and invasion by the activation of MEK/ERK pathway. Transcriptome sequencing analysis revealed that CST1 is closely associated with OXPHOS. Based on a real-time ATP rate assay, mitochondrial complex I enzyme activity assay, immunofluorescence, co-immunoprecipitation, and addition of the OXPHOS inhibitor Rotenone and MEK/ERK inhibitor PD98059, we determined that CST1 affects mitochondrial complex I enzyme activity by interacting with the GRIM19 protein to elevate OXPHOS levels, and a reciprocal regulatory relationship exists between OXPHOS and the MEK/ERK pathway in ESCC cells. Finally, an in vivo study demonstrated the potential of CST1 in ESCC metastasis through regulation of the OXPHOS and MEK/ERK pathways. This study is the first to reveal the oncogenic role of CST1 in ESCC development by enhancing mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis, and then promote ESCC metastasis, suggesting that CST1/OXPHOS is a promising target for ESCC treatment.


Sujet(s)
Diazo-méthane/analogues et dérivés , Dipeptides , Tumeurs de l'oesophage , Carcinome épidermoïde de l'oesophage , Humains , Carcinome épidermoïde de l'oesophage/métabolisme , Tumeurs de l'oesophage/anatomopathologie , Phosphorylation oxydative , Projets pilotes , Mitogen-Activated Protein Kinase Kinases/métabolisme , Lignée cellulaire tumorale , Régulation de l'expression des gènes tumoraux , Prolifération cellulaire , Mouvement cellulaire
2.
Org Biomol Chem ; 20(35): 7027-7030, 2022 09 14.
Article de Anglais | MEDLINE | ID: mdl-36017814

RÉSUMÉ

We have developed a convenient synthesis of a series of ß-fluoramides in 65% yield. The process involved a tandem fluorination/Ritter reaction to synthesize ß-fluoramides using α-diazo 2H-benzopyran-4-one compounds. Selectfluor was used as the electrophilic fluoride source in acetonitrile to build the ß-fluorinated quaternary carbon center and amide derivatives of 2H-benzopyran-4-one in one step. The products N-(2-(2-fluoro-2,3-dihydro-3-oxobenzofuran-2-yl)propan-2-yl)acetamides were a series of bifunctional compounds with a 2-fluoro-2,3-dihydro-3-oxobenzofuran motif and amide groups.


Sujet(s)
Acétamides , Benzopyranes , Diazo-méthane/analogues et dérivés , Acide egtazique/analogues et dérivés , Structure moléculaire
3.
ACS Appl Mater Interfaces ; 13(31): 36839-36848, 2021 Aug 11.
Article de Anglais | MEDLINE | ID: mdl-34342218

RÉSUMÉ

Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate diazirine, despite having little to no absorption at 445 nm. CaproGlu's liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity.


Sujet(s)
Adhésifs/composition chimique , Matériaux biocompatibles/composition chimique , Complexes de coordination/composition chimique , Diazo-méthane/analogues et dérivés , Animaux , Catalyse/effets des radiations , Collagène/composition chimique , Complexes de coordination/effets des radiations , Complexes de coordination/toxicité , Réactifs réticulants/composition chimique , Iridium/composition chimique , Iridium/effets des radiations , Iridium/toxicité , Lumière , Souris , Cellules NIH 3T3 , Polyesters/composition chimique , Suidae
4.
ACS Chem Biol ; 16(8): 1493-1507, 2021 08 20.
Article de Anglais | MEDLINE | ID: mdl-34355883

RÉSUMÉ

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.


Sujet(s)
Cholestérol/analogues et dérivés , Diazo-méthane/analogues et dérivés , Canaux ioniques régulés par des ligands/composition chimique , Marqueurs de photoaffinité/composition chimique , Alcynes/synthèse chimique , Alcynes/composition chimique , Alcynes/métabolisme , Sites de fixation , Cholestérol/synthèse chimique , Cholestérol/métabolisme , Cyanobactéries/composition chimique , Diazo-méthane/synthèse chimique , Diazo-méthane/métabolisme , Colorants fluorescents/composition chimique , Canaux ioniques régulés par des ligands/métabolisme , Simulation de docking moléculaire , Simulation de dynamique moléculaire , Marqueurs de photoaffinité/synthèse chimique , Marqueurs de photoaffinité/métabolisme , Liaison aux protéines
5.
Methods Mol Biol ; 2306: 77-91, 2021.
Article de Anglais | MEDLINE | ID: mdl-33954941

RÉSUMÉ

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.


Sujet(s)
Lipidomique/méthodes , Lysophospholipides/analyse , Monoglycérides/analyse , Phosphatidylglycérol/analyse , Phosphates phosphatidylinositol/analyse , Animaux , Diazo-méthane/analogues et dérivés , Diazo-méthane/composition chimique , Tests de criblage à haut débit , Humains , Isomérie , Lysophospholipides/composition chimique , Méthylation , Souris , Monoglycérides/composition chimique , Phosphatidylglycérol/composition chimique , Phosphates phosphatidylinositol/composition chimique , Spectrométrie de masse ESI , Composés triméthylsilyl/composition chimique
6.
Mol Cell Proteomics ; 20: 100084, 2021.
Article de Anglais | MEDLINE | ID: mdl-33915260

RÉSUMÉ

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.


Sujet(s)
Réactifs réticulants/composition chimique , Diazo-méthane/analogues et dérivés , Diazo-méthane/composition chimique , Chromatographie en phase liquide , Protéines fongiques/composition chimique , Spectrométrie de masse/méthodes , Processus photochimiques , Proteasome endopeptidase complex/composition chimique , Saccharomyces cerevisiae , Sérumalbumine bovine
7.
ACS Chem Biol ; 16(2): 404-413, 2021 02 19.
Article de Anglais | MEDLINE | ID: mdl-33543920

RÉSUMÉ

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.


Sujet(s)
Marqueurs d'affinité/composition chimique , Azotures/composition chimique , Réactifs réticulants/composition chimique , Diazo-méthane/analogues et dérivés , Hydrocarbures chlorés/composition chimique , Protéomique/méthodes , Marqueurs d'affinité/effets des radiations , Azotures/effets des radiations , Chromatographie en phase liquide , Réactifs réticulants/effets des radiations , Dasatinib/analogues et dérivés , Dasatinib/pharmacologie , Dasatinib/effets des radiations , Diazo-méthane/effets des radiations , Histone deacetylases/analyse , Histone deacetylases/composition chimique , Humains , Hydrocarbures chlorés/effets des radiations , Hydrolases/composition chimique , Cellules K562 , Spectrométrie de masse , Propranolol/analogues et dérivés , Propranolol/pharmacologie , Propranolol/effets des radiations , Protein kinases/analyse , Protein kinases/composition chimique , Récepteurs alpha-2 adrénergiques/analyse , Récepteurs alpha-2 adrénergiques/composition chimique , Rayons ultraviolets , Vorinostat/analogues et dérivés , Vorinostat/pharmacologie , Vorinostat/effets des radiations
8.
ACS Chem Biol ; 16(2): 389-396, 2021 02 19.
Article de Anglais | MEDLINE | ID: mdl-33524253

RÉSUMÉ

Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD+ enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD+ provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.


Sujet(s)
Réactifs réticulants/métabolisme , NAD/métabolisme , Poly (ADP-Ribose) polymerase-1/métabolisme , Protéome/métabolisme , Azotures/synthèse chimique , Azotures/métabolisme , Azotures/effets des radiations , Chimie click , Réactifs réticulants/synthèse chimique , Réactifs réticulants/effets des radiations , Diazo-méthane/analogues et dérivés , Diazo-méthane/métabolisme , Diazo-méthane/effets des radiations , Cellules HEK293 , Humains , NAD/synthèse chimique , NAD/effets des radiations , Poly(ADP-ribosylation) , Maturation post-traductionnelle des protéines , Protéome/composition chimique , Protéomique , Rayons ultraviolets
9.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Article de Anglais | MEDLINE | ID: mdl-33468658

RÉSUMÉ

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.


Sujet(s)
Diazo-méthane/métabolisme , Histone/génétique , Lysine/métabolisme , Cadres ouverts de lecture , Peptides/génétique , Transcription génétique , Séquence d'acides aminés , Animaux , Bovins , Chromatine/composition chimique , Chromatine/métabolisme , Diazo-méthane/analogues et dérivés , Régulation de l'expression des gènes , Locus génétiques , Cellules HEK293 , Cellules HeLa , Histone/métabolisme , Humains , Cellules K562 , Lysine/analogues et dérivés , Souris , Pan troglodytes , Peptides/métabolisme , Liaison aux protéines/effets des radiations , Cartographie d'interactions entre protéines , Rats , Alignement de séquences , Similitude de séquences d'acides aminés , Transcription génétique/effets des radiations , Transgènes , Rayons ultraviolets
10.
Meat Sci ; 172: 108339, 2021 Feb.
Article de Anglais | MEDLINE | ID: mdl-33171309

RÉSUMÉ

This study examines the effects of ageing (1, 14 days), cathepsin inhibition (No or Yes) and temperature (25-90 °C) on the shrinkage of fibre fragments from three bovine muscles (semitendinosus, biceps femoris and psoas major) during heating. Shrinkage was quantified using light microscopy images. Muscle fibres (except in psoas major) had greater transverse shrinkage, and less longitudinal shrinkage in aged than in unaged muscles at temperatures ≥60-75 °C. In addition, cathepsin inhibition during heating at ≥65-90 °C caused greater transverse shrinkage in semitendinosus fibres, and reduced longitudinal shrinkage for all muscles. At temperatures ≥75 °C, the longitudinal and transverse shrinkage of the fibres was correlated for all muscles. Ageing of biceps femoris increases volume shrinkage on a fibre level, and hence potentially cooking loss, while cathepsin activity in the semitendinosus reduces volume shrinkage. In conclusion, cathepsin activity and ageing influence the shrinkage that occurs during heating and these factors should be explored further to enable optimisation of thermal meat processing.


Sujet(s)
Cathepsines/métabolisme , Cuisine (activité) , Fibres musculaires squelettiques , Viande rouge/analyse , Animaux , Bovins , Diazo-méthane/analogues et dérivés , Diazo-méthane/pharmacologie , Facteurs temps
11.
ACS Chem Biol ; 15(9): 2374-2381, 2020 09 18.
Article de Anglais | MEDLINE | ID: mdl-32804474

RÉSUMÉ

RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.


Sujet(s)
Azotures/composition chimique , Diazo-méthane/analogues et dérivés , Marqueurs de photoaffinité/composition chimique , ARN/métabolisme , Aptamères nucléotidiques/composition chimique , Aptamères nucléotidiques/métabolisme , Azotures/métabolisme , Azotures/effets des radiations , Sites de fixation , Diazo-méthane/métabolisme , Diazo-méthane/effets des radiations , Ligands , Simulation de docking moléculaire , Marqueurs de photoaffinité/métabolisme , Marqueurs de photoaffinité/effets des radiations , Étude de validation de principe , ARN/composition chimique , Transcription inverse , Analyse de séquence d'ADN
12.
Chembiochem ; 21(7): 924-932, 2020 04 01.
Article de Anglais | MEDLINE | ID: mdl-31794116

RÉSUMÉ

Studying protein-protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells.


Sujet(s)
Réactifs réticulants/composition chimique , Protéines/composition chimique , Rayons ultraviolets , Toxine cholérique/composition chimique , Réactifs réticulants/métabolisme , Diazo-méthane/analogues et dérivés , Diazo-méthane/composition chimique , Humains , Ligases/métabolisme , Lysine/analogues et dérivés , Lysine/composition chimique , Maturation post-traductionnelle des protéines , Protéines/métabolisme
13.
Biochem Biophys Res Commun ; 512(1): 100-105, 2019 04 23.
Article de Anglais | MEDLINE | ID: mdl-30871775

RÉSUMÉ

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.


Sujet(s)
Protéines Escherichia coli/composition chimique , Motifs et domaines d'intéraction protéique , Benzophénones/composition chimique , Benzophénones/métabolisme , Réactifs réticulants , Diazo-méthane/analogues et dérivés , Diazo-méthane/composition chimique , Diazo-méthane/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Protéines Escherichia coli/génétique , Protéines Escherichia coli/métabolisme , Protéines du choc thermique/composition chimique , Protéines du choc thermique/génétique , Protéines du choc thermique/métabolisme , Lysine/analogues et dérivés , Lysine/composition chimique , Lysine/métabolisme , Modèles moléculaires , Simulation de dynamique moléculaire , Mutation , Protéines périplasmiques/composition chimique , Protéines périplasmiques/génétique , Protéines périplasmiques/métabolisme , Phénylalanine/analogues et dérivés , Phénylalanine/composition chimique , Phénylalanine/métabolisme , Multimérisation de protéines , Structure quaternaire des protéines , Sous-unités de protéines , Serine endopeptidases/composition chimique , Serine endopeptidases/génétique , Serine endopeptidases/métabolisme
14.
Mol Pharm ; 16(3): 1053-1064, 2019 03 04.
Article de Anglais | MEDLINE | ID: mdl-30721080

RÉSUMÉ

Interactions of a lyophilized peptide with water and excipients in a solid matrix were explored using photolytic labeling. A model peptide "KLQ" (Ac-QELHKLQ-NHCH3) was covalently labeled with NHS-diazirine (succinimidyl 4,4'-azipentanoate), and the labeled peptide (KLQ-SDA) was formulated and exposed to UV light in both solution and lyophilized solids. Solid samples contained the following excipients at a 1:400 molar ratio: sucrose, trehalose, mannitol, histidine, or arginine. Prior to UV exposure, the lyophilized solids were exposed to various relative humidity (RH) environments (8, 13, 33, 45, and 78%), and the resulting solid moisture content (Karl Fischer titration) and glass transition temperature ( Tg; differential scanning calorimetry, DSC) were measured. To initiate photolytic labeling, solution and solid samples were exposed to UV light at 365 nm for 30 min. Photolytic-labeling products were quantified using reversed-phase high-performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In lyophilized solids, studies excluding oxygen and using H218O confirmed that the source of oxygen in KLQ adducts with a mass increase of 18 amu are attributable to reaction with water, while those with a mass increase of 16 amu are not attributable to reaction with either water or molecular oxygen. In solids containing sucrose or trehalose, peptide-excipient adducts decreased with increasing solid moisture content, while peptide-water adducts increased only at lower RH exposure and then plateaued, in partial agreement with the water replacement hypothesis.


Sujet(s)
Chimie pharmaceutique/méthodes , Préparation de médicament/méthodes , Lyophilisation/méthodes , Peptides/composition chimique , Photolyse/effets des radiations , Eau/composition chimique , Calorimétrie différentielle à balayage , Chromatographie en phase inverse , Diazo-méthane/analogues et dérivés , Diazo-méthane/composition chimique , Excipients/composition chimique , Humidité , Liaison hydrogène , Spectrométrie de masse , Oxygène/composition chimique , Saccharose/composition chimique , Température de transition , Tréhalose/composition chimique , Rayons ultraviolets , Vitrification
15.
Anal Chem ; 91(1): 1019-1026, 2019 01 02.
Article de Anglais | MEDLINE | ID: mdl-30525455

RÉSUMÉ

Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C18 column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.


Sujet(s)
Désoxyribonucléotides/analyse , Diazo-méthane/composition chimique , Ribonucléotides/analyse , Cellules A549 , Chromatographie en phase liquide , Diazo-méthane/analogues et dérivés , Cellules HCT116 , Humains , Spectrométrie de masse en tandem , Cellules cancéreuses en culture
16.
ACS Chem Biol ; 13(12): 3333-3342, 2018 12 21.
Article de Anglais | MEDLINE | ID: mdl-30489064

RÉSUMÉ

Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.


Sujet(s)
Marqueurs d'affinité/composition chimique , Aspartic acid endopeptidases/composition chimique , Diazo-méthane/analogues et dérivés , Gelatinases/composition chimique , Glutamate carboxypeptidase II/composition chimique , Protéines membranaires/composition chimique , Protéomique/méthodes , Serine endopeptidases/composition chimique , Marqueurs d'affinité/synthèse chimique , Marqueurs d'affinité/effets des radiations , Aspartic acid endopeptidases/antagonistes et inhibiteurs , Biotine/composition chimique , Lignée cellulaire tumorale , Diazo-méthane/synthèse chimique , Diazo-méthane/effets des radiations , Endopeptidases , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Antienzymes/effets des radiations , Fluorescéines/composition chimique , Colorants fluorescents/composition chimique , Gelatinases/antagonistes et inhibiteurs , Glutamate carboxypeptidase II/antagonistes et inhibiteurs , Humains , Spectrométrie de masse/méthodes , Protéines membranaires/antagonistes et inhibiteurs , Microscopie confocale/méthodes , Poly(acides méthacryliques)/composition chimique , Rayons ultraviolets
17.
Anal Chim Acta ; 1024: 101-111, 2018 Sep 18.
Article de Anglais | MEDLINE | ID: mdl-29776536

RÉSUMÉ

In this study, we developed a novel strategy using solid-phase extraction (SPE) coupled with shotgun mass spectrometry (MS) based on trimethylsilyldiazomethane (TMSCHN2) stable-isotope derivatization for rapid profiling and accurate quantification of phospholipids (PLs) in human plasma. HybridSPE-Phospholipid (HybridSPE-PL, zirconia coated silica stationary phase) was used for sample pretreatment via the Lewis acid-base interaction between zirconia and phosphate moiety of PLs. This step allows rapid enrichment and recovery of PLs from human plasma. Afterward, PLs were derivatized with TMSCHN2, which leads to methylation of hydroxyl and amino groups in PLs and allows highly sensitive PL analysis by shotgun MS in positive ionization mode (limit of detection decreased up to 116.67 fold compared to underived PLs). We developed an accuracy quantification method for determination of PL molecular species in biological samples. Two or more PL standards were selected for each PL class and derivatized with TMSCHN2 without stable-isotope coding. They were then used as the internal standards. PLs in biological samples were isotopic derivatized via acid-catalyzed H/D exchange and methanolysis of TMSCHN2. For accurate quantification, a calibration curve for each class of PLs was typically constructed by using the internal standards to normalize the non-uniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual PL species in the biological samples. This newly developed method was used to comprehensively analyze PL molecular species in human plasma samples. It is a promising methodology for rapid profiling and accurate quantification of complex lipid molecules in biological samples.


Sujet(s)
Diazo-méthane/analogues et dérivés , Phospholipides/sang , Plasma sanguin/composition chimique , Spectrométrie de masse ESI , Composés triméthylsilyl/composition chimique , Calibrage , Diazo-méthane/composition chimique , Humains , Marquage isotopique , Cinétique , Limite de détection , Méthylation , Phospholipides/composition chimique , Recherche qualitative , Silice/composition chimique , Extraction en phase solide , Zirconium/composition chimique
18.
Chembiochem ; 19(15): 1638-1642, 2018 08 06.
Article de Anglais | MEDLINE | ID: mdl-29732707

RÉSUMÉ

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next-generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane.


Sujet(s)
Antinéoplasiques alcoylants/pharmacologie , ADN/composition chimique , Diazo-méthane/analogues et dérivés , ARN/composition chimique , Composés triméthylsilyl/pharmacologie , Alkylation/effets des médicaments et des substances chimiques , Antinéoplasiques alcoylants/composition chimique , Diazo-méthane/composition chimique , Diazo-méthane/pharmacologie , Séquençage nucléotidique à haut débit , Humains , Tumeurs/traitement médicamenteux , Composés triméthylsilyl/composition chimique
19.
Leg Med (Tokyo) ; 30: 14-20, 2018 Jan.
Article de Anglais | MEDLINE | ID: mdl-29127845

RÉSUMÉ

A highly sensitive method was developed for the analysis of alendronate in human plasma and dialysate using MonoSpin™ SAX® extraction and metal-free high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) following methylation with trimethylsilyldiazomethane. The chromatographic separation of the derivatives for alendronate and alendronate-d6 was achieved on an L-column2 ODS metal-free column (50 mm  ×  2 mm i.d., particle size 3 µm) with a linear gradient elution system composed of 10 mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.3 ml/min. Quantification was performed by multiple reaction monitoring (MRM) with positive-ion electrospray ionization (ESI). Distinct peaks were observed for alendronate and for the internal standard on each channel within 1 min. The regression equations showed good linearity within the ranges of 2.0-100 ng/0.5 ml for the plasma and 1.0-100 ng/0.5 ml for the dialysate, with the limits of detection at 1.0 ng/0.5 ml for the plasma and 0.5 ng/0.5 ml for the dialysate. Extraction efficiencies for alendronate for the plasma and dialysate were 41.1-51.2% and 63.6-73.4%, respectively. The coefficient of variation (CV) was ≤8.5%. The method was successfully applied to the analyses of real plasma and dialysate samples derived after intravenous administration of alendronate.


Sujet(s)
Alendronate/sang , Agents de maintien de la densité osseuse/sang , Chromatographie en phase liquide à haute performance/méthodes , Solutions de dialyse/analyse , Plasma sanguin/composition chimique , Spectrométrie de masse en tandem/méthodes , Diazo-méthane/analogues et dérivés , Humains , Métaux , Composés triméthylsilyl
20.
Acc Chem Res ; 50(5): 1184-1192, 2017 05 16.
Article de Anglais | MEDLINE | ID: mdl-28467057

RÉSUMÉ

The cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host-pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein-protein interactions under living conditions. Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein-protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking. Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone-client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein-protein interactions within bacterial cell envelope is also discussed.


Sujet(s)
Protéines Escherichia coli/métabolisme , Chaperons moléculaires/métabolisme , Réactifs réticulants/effets des radiations , Diazo-méthane/analogues et dérivés , Diazo-méthane/effets des radiations , Escherichia coli/composition chimique , Escherichia coli/métabolisme , Protéines Escherichia coli/composition chimique , Protéines Escherichia coli/génétique , Concentration en ions d'hydrogène , Lysine/analogues et dérivés , Lysine/génétique , Lysine/effets des radiations , Chaperons moléculaires/composition chimique , Chaperons moléculaires/génétique , Marqueurs de photoaffinité/effets des radiations , Liaison aux protéines , Ingénierie des protéines , Cartographie d'interactions entre protéines
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