Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
1.
Nature ; 626(8001): 1141-1148, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38326620

RESUMEN

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.


Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Receptores Sensibles al Calcio , Humanos , Regulación Alostérica/efectos de los fármacos , Cinacalcet/farmacología , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ligandos , Lípidos , Nanoestructuras/química , Poliaminas/metabolismo , Conformación Proteica/efectos de los fármacos , Receptores Sensibles al Calcio/química , Receptores Sensibles al Calcio/metabolismo , Receptores Sensibles al Calcio/ultraestructura , Especificidad por Sustrato , Triptófano/metabolismo , Calcio/metabolismo
2.
Anal Chem ; 96(35): 14186-14196, 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39171919

RESUMEN

Protein S-sulfhydration involves the regulation of various protein functions, and resolving the S-sulfhydrated proteome (persulfidome) allows for a deeper exploration of various redox regulations. Therefore, we designed a reducible covalent capture method for isolating S-sulfhydrated proteins, which can analyze the persulfidome in biological samples and monitor specific S-sulfhydrated proteins. In this study, we applied this method to reveal the S-sulfhydration levels of proteins, including 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin. Furthermore, this technique can be used to enrich S-sulfhydrated peptides, aiding in the determination of protein S-sulfhydration modification sites. Finally, we observed that the S-sulfhydration and oxidation of nucleolin on the C543 residue correlate with its nuclear translocation, downstream regulation of p53, Bcl-xL, and Bcl-2 RNA levels and protein expression, as well as the protective function against oxidative stress. Therefore, this method may facilitate the understanding of the regulation of protein function by redox perturbation.


Asunto(s)
Nucleolina , Oxidación-Reducción , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/análisis , Humanos , Proteoma/análisis , Proteoma/química
3.
Drug Metab Rev ; : 1-38, 2024 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-38963129

RESUMEN

Advances in the field of bioactivation have significantly contributed to our understanding and prediction of drug-induced liver injury (DILI). It has been established that many adverse drug reactions, including DILI, are associated with the formation and reactivity of metabolites. Modern methods allow us to detect and characterize these reactive metabolites in earlier stages of drug development, which helps anticipate and circumvent the potential for DILI. Improved in silico models and experimental techniques that better reflect in vivo environments are enhancing predictive capabilities for DILI risk. Further, studies on the mechanisms of bioactivation, including enzyme interactions and the role of individual genetic differences, have provided valuable insights for drug optimizations. Cumulatively, this progress is continually refining our approaches to drug safety evaluation and personalized medicine.

4.
Anal Chem ; 95(41): 15245-15253, 2023 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-37791746

RESUMEN

Top-down proteomics, the tandem mass spectrometric analysis of intact proteoforms, is the dominant method for proteoform characterization in complex mixtures. While this strategy produces detailed molecular information, it also requires extensive instrument time per mass spectrum obtained and thus compromises the depth of proteoform coverage that is accessible on liquid chromatography time scales. Such a top-down analysis is necessary for making original proteoform identifications, but once a proteoform has been confidently identified, the extensive characterization it provides may no longer be required for a subsequent identification of the same proteoform. We present a strategy to identify proteoforms in tissue samples on the basis of the combination of an intact mass determination with a measured count of the number of cysteine residues present in each proteoform. We developed and characterized a cysteine tagging chemistry suitable for the efficient and specific labeling of cysteine residues within intact proteoforms and for providing a count of the cysteine amino acids present. On simple protein mixtures, the tagging chemistry yields greater than 98% labeling of all cysteine residues, with a labeling specificity of greater than 95%. Similar results are observed on more complex samples. In a proof-of-principle study, proteoforms present in a human prostate tumor biopsy were characterized. Observed proteoforms, each characterized by an intact mass and a cysteine count, were grouped into proteoform families (groups of proteoforms originating from the same gene). We observed 2190 unique experimental proteoforms, 703 of which were grouped into 275 proteoform families.


Asunto(s)
Cisteína , Espectrometría de Masas en Tándem , Humanos , Cisteína/metabolismo , Espectrometría de Masas en Tándem/métodos , Proteínas/metabolismo , Cromatografía Liquida , Proteómica/métodos , Proteoma/análisis , Procesamiento Proteico-Postraduccional
5.
Anal Chem ; 95(50): 18504-18513, 2023 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-38033201

RESUMEN

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.


Asunto(s)
Aminoácidos , Proteómica , Animales , Ratones , Aminoácidos/análisis , Proteómica/métodos , Leucina/química , Aminas , Cromatografía Liquida/métodos
6.
Proteomics ; 22(19-20): e2100256, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35687565

RESUMEN

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.


Asunto(s)
Proteoma , Proteómica , Proteómica/métodos , Proteoma/metabolismo , Espectrometría de Masas en Tándem/métodos , Marcaje Isotópico/métodos , Péptidos/análisis , Biomarcadores , Lípidos
7.
Anal Chem ; 94(38): 13036-13042, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36099193

RESUMEN

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.


Asunto(s)
Ácidos Grasos , Espectrometría de Masas en Tándem , Aldehídos , Carbono , Ácidos Carboxílicos , Clorobenzoatos , Ácidos Grasos/química , Hidrazinas , Indicadores y Reactivos , Cetonas , Polisacáridos/análisis , Espectrometría de Masas en Tándem/métodos
8.
Anal Chem ; 94(9): 3774-3781, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35189681

RESUMEN

Many metabolites, including amino acids, neurotransmitters, and pharmaceuticals, contain primary amine functional groups. The analysis of these molecules by mass spectrometry (MS) plays an important role in the study of cancers and psychogenic diseases. However, the MS-based detection and visualization of these bioactive metabolites directly from real biological systems still suffer from challenges such as low ionization efficiency and/or matrix interference effects. Here, we introduce a simple and efficient strategy, the nanosecond photochemical reaction (nsPCR)-enabled fast chemical derivatization, enabling direct MS analysis of primary amine-containing metabolites, with enhanced detection sensitivity for numerous metabolites from cell culture medium and rat brain sections. Furthermore, this nsPCR-based chemical derivatization strategy was demonstrated to be a useful visualizing tool that could provide improved spatial information for these metabolites, potentially offering alternative tools for gaining novel insights into metabolic events.


Asunto(s)
Aminas , Aminoácidos , Aminas/química , Aminoácidos/análisis , Animales , Indicadores y Reactivos , Neurotransmisores , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
9.
Anal Chem ; 94(34): 11773-11782, 2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-35960654

RESUMEN

Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass spectrometry (MS)-based glycoproteomic analysis is hindered by the low abundance and poor ionization efficiency of glycopeptides. Relatively large amounts of starting materials are needed for the enrichment, which makes the identification and quantification of intact glycopeptides from samples with limited quantity more challenging. To overcome these limitations, we developed an improved isobaric labeling strategy with an additional boosting channel to enhance N,N-dimethyl leucine (DiLeu) tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With the integration of a one-tube sample processing workflow and high-pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 µg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal fluid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer's disease (AD) patients and non-AD donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, weighted gene coexpression network analysis (WGCNA) showed nine modules of glycopeptides, two of which were associated with the AD phenotype. Our results demonstrated the feasibility of using this strategy for in-depth glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for the enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems with a limited sample quantity.


Asunto(s)
Glicopéptidos , Glicopéptidos/análisis , Células HeLa , Humanos , Leucina/análogos & derivados , Leucina/química , Espectrometría de Masas
10.
Anal Chem ; 93(28): 9845-9852, 2021 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-34240851

RESUMEN

As one of the most important post-translational modifications, glycosylation plays a pivotal role in many essential physiological functions, including cell recognition, signaling, and immune response. Thus, various qualitative and quantitative analytical strategies for glycomic profiling have been developed in recent decades. However, while extensive efforts have been devoted to the analysis of N-glycans, high-throughput quantitative analysis of O-glycans is often overlooked and underexplored. This is partially due to the lack of a universal enzyme for the release of O-glycans from the protein backbone. Furthermore, the traditional chemical releasing method suffers from severe side reactions and involves tedious sample preparation procedures. Here, a multiplexed isobaric labeling method enabled by N,N-dimethyl leucine containing pyrazolone analogue (DiLeuPMP) is introduced. This method combines the release and labeling of O-glycans in a one-pot reaction and achieves accurate MS2-based relative quantification with the ability to process four samples at a time. The method has been applied to core-1 O-glycan standard and three glycoproteins first, and the results demonstrated its validity. Following this proof-of-principle demonstration, we analyzed more complex biological specimen using human serum samples. Overall, this method provides an effective and reliable approach for the profiling and high-throughput quantitative analysis of O-glycans in complex samples.


Asunto(s)
Polisacáridos , Espectrometría de Masa por Ionización de Electrospray , Glicoproteínas , Glicosilación , Humanos , Procesamiento Proteico-Postraduccional
11.
Anal Chem ; 92(16): 11119-11126, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32649829

RESUMEN

The unbiased selection of peptide precursors makes data-independent acquisition (DIA) an advantageous alternative to data-dependent acquisition (DDA) for discovery proteomics, but traditional multiplexed quantification approaches employing mass difference labeling or isobaric tagging are incompatible with DIA. Here, we describe a strategy that permits multiplexed quantification by DIA using mass defect-based N,N-dimethyl leucine (mdDiLeu) tags and high-resolution tandem mass spectrometry (MS2) analysis. Millidalton mass differences between mdDiLeu isotopologues produce fragment ion multiplet peaks separated in mass by as little as 5.8 mDa, enabling up to 4-plex quantification in DIA MS2 spectra. Quantitative analysis of yeast samples displayed comparable accuracy and precision for MS2-based DIA and MS1-based DDA methods. Multiplexed DIA analysis of cerebrospinal fluid revealed the dynamic proteome changes in Alzheimer's disease, demonstrating its utility for discovery of potential clinical biomarkers. We show that the mdDiLeu tagging approach for multiplexed DIA is a viable methodology for investigating proteome changes, particularly for low-abundance proteins, in different biological matrices.


Asunto(s)
Leucina/análogos & derivados , Proteoma/análisis , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secuencia de Aminoácidos , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/química , Proteínas del Líquido Cefalorraquídeo/líquido cefalorraquídeo , Proteínas del Líquido Cefalorraquídeo/química , Humanos , Persona de Mediana Edad , Prueba de Estudio Conceptual , Proteoma/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Masas en Tándem
12.
Chemistry ; 24(7): 1522-1527, 2018 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-29226433

RESUMEN

[2]Rotaxanes displaying one-off photo-triggerable gelation properties have been synthesized through the "clipping" of photo-degradable macrocycles around the amide or urea functionalities of organo- and hydrogelators. Irradiation with UV-light cleaved the photo-labile macrocyclic components from the [2]rotaxanes, resulting in the free gelators being released into solution and, thereafter, forming gels. When the rate of gelation was sufficiently rapid, selective gelation of specific regions of the solution-and, indeed, photo-patterning of the solution-was possible.

13.
Nat Chem ; 16(5): 762-770, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38365942

RESUMEN

Mass spectrometry-based quantitative lipidomics is an emerging field aiming to uncover the intricate relationships between lipidomes and disease development. However, quantifying lipidomes comprehensively in a high-throughput manner remains challenging owing to the diverse lipid structures. Here we propose a diazobutanone-assisted isobaric labelling strategy as a rapid and robust platform for multiplexed quantitative lipidomics across a broad range of lipid classes, including various phospholipids and glycolipids. The diazobutanone reagent is designed to conjugate with phosphodiester or sulfate groups, while accommodating various functional groups on different lipid classes, enabling subsequent isobaric labelling for high-throughput multiplexed quantitation. Our method demonstrates excellent performance in terms of labelling efficiency, detection sensitivity, quantitative accuracy and broad applicability to various biological samples. Finally, we performed a six-plex quantification analysis of lipid extracts from lean and obese mouse livers. In total, we identified and quantified 246 phospholipids in a high-throughput manner, revealing lipidomic changes that may be associated with obesity in mice.


Asunto(s)
Glucolípidos , Lipidómica , Fosfolípidos , Espectrometría de Masas en Tándem , Animales , Glucolípidos/química , Fosfolípidos/química , Lipidómica/métodos , Espectrometría de Masas en Tándem/métodos , Ratones , Sulfatos/química , Hígado/metabolismo , Hígado/química
14.
ACS Appl Mater Interfaces ; 15(41): 47893-47901, 2023 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-37812448

RESUMEN

Protein phosphorylation is an important post-translational modification (PTM), which is involved in many important cellular functions. Understanding protein phosphorylation at the molecular level is critical to deciphering its relevant biological processes and signaling networks. Mass spectrometry (MS) has become a powerful tool for the comprehensive profiling of protein phosphorylation. Yet the low ionization efficiency and low abundance of phosphopeptides among complex biological samples make its MS analysis challenging; an enrichment strategy with high efficiency and selectivity is always necessary prior to MS analysis. In this study, we developed a phosphorylated cotton-fiber-based Ti(IV)-IMAC material (termed as Cotton Ti-IMAC) that can serve as a novel platform for phosphopeptide enrichment. The cotton fiber can be effectively grafted with phosphate groups covalently in a single step, where the titanium ions can then be immobilized to enable capturing phosphopeptides. The material can be prepared using cost-effective reagents within only 4 h. Benefiting from the flexibility and filterability of cotton fibers, the material can be easily packed as a spin-tip and make the enrichment process convenient. Cotton Ti-IMAC successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (BSA/ß-casein = 1000:1) and excellent sensitivity (0.1 fmol/µL). Moreover, 2354 phosphopeptides were profiled in one LC-MS/MS injection after enriching from only 100 µg of HeLa cell digests with an enrichment specificity of up to 97.51%. Taken together, we believe that Cotton Ti-IMAC can serve as a widely applicable and robust platform for achieving large-scale phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex biological systems.


Asunto(s)
Fosfopéptidos , Titanio , Humanos , Titanio/química , Células HeLa , Fosfopéptidos/química , Cromatografía Liquida , Espectrometría de Masas en Tándem , Cromatografía de Afinidad/métodos
15.
Anal Chim Acta ; 1225: 340215, 2022 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-36038233

RESUMEN

Fatty acids (FAs) possess highly diverse structures and can be divided into saturated and unsaturated classes. For unsaturated FAs, both the numbers and positions of carbon-carbon double bond (C=C) determine their biological functions. Abnormal levels of FA isomers have been reported to be involved in various disease development, such as cancer. Despite numerous advances in lipidomics, simultaneous quantifying and pinpointing C=C bond positions in a high-throughput manner remains a challenge. Here we conducted epoxidation of C=C bonds of unsaturated FAs followed by the conjugation with isobaric SUGAR tags. With the assistance of LC-MS, FA isomers with the same masses were separated on the C18 column and individually subjected to MS/MS fragmentation. Upon higher-energy collisional dissociation, not only reporter ions for multiplexed quantification but also diagnostic ions for C=C localization were generated at the same time, allowing quantitative analyses of different unsaturated FA isomers in samples. The performance of this approach including epoxidation, labeling efficiencies, quantitation accuracy, and capability to pinpoint C=C bond position were evaluated. To evaluate our method, free FA extracts from healthy human serum were used to demonstrate the feasibility of this method for complex sample analysis. Finally, this method was also applied to investigate the changes of unsaturated FA isomers between heathy human and Alzheimer's disease (AD) patient serum.


Asunto(s)
Ácidos Grasos , Espectrometría de Masas en Tándem , Carbono , Cromatografía Liquida , Ácidos Grasos Insaturados/análisis , Humanos , Indicadores y Reactivos , Iones , Compuestos Orgánicos , Espectrometría de Masas en Tándem/métodos
16.
J Chromatogr A ; 1552: 43-52, 2018 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-29650481

RESUMEN

Hydrogen sulfide (H2S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H2S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H2S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). 13C6-Labeled and label-free 2-IAN efficiently react with H2S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of H2S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13C6-labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 µM linear ranges for DL-homocysteine and glutathione and 1 nM-1 µM linear ranges for l-cysteine and H2S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13C6-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis.


Asunto(s)
Acetanilidas/química , Sulfuro de Hidrógeno/análisis , Espectrometría de Masa por Ionización de Electrospray , Compuestos de Sulfhidrilo/análisis , Isótopos de Carbono/química , Cromatografía Líquida de Alta Presión , Cisteína/análisis , Glutatión/análisis , Células Hep G2 , Homocisteína/análisis , Humanos , Sulfuro de Hidrógeno/metabolismo , Marcaje Isotópico , Compuestos de Sulfhidrilo/metabolismo
17.
Nat Commun ; 8(1): 2272, 2017 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-29273778

RESUMEN

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Dominio Catalítico , Cristalización , Células HEK293 , Holoenzimas , Humanos , Metilación , Ratones , Chaperonas Moleculares , Transducción de Señal
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA