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1.
J Am Chem Soc ; 146(17): 11702-11710, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38640258

RESUMO

Sulfuric acid in the atmosphere can participate in acid-catalyzed and acid-driven reactions, including those within secondary organic aerosols (SOA). Previous studies have observed enhanced absorption at visible wavelengths and significant changes in the chemical composition when SOA was exposed to sulfuric acid. However, the specific chromophores responsible for these changes could not be identified. The goals of this study are to identify the chromophores and determine the mechanism of browning in highly acidified α-pinene SOA by following the behavior of specific common α-pinene oxidation products, namely, cis-pinonic acid and cis-pinonaldehyde, when they are exposed to highly acidic conditions. The products of these reactions were analyzed with ultra-performance liquid chromatography coupled with photodiode array spectrophotometry and high-resolution mass spectrometry, UV-vis spectrophotometry, and nuclear magnetic resonance spectroscopy. cis-Pinonic acid (2) was found to form homoterpenyl methyl ketone (4), which does not absorb visible radiation, while cis-pinonaldehyde (3) formed weakly absorbing 1-(4-(propan-2-ylidene)cyclopent-1-en-1-yl)ethan-1-one (5) and 1-(4-isopropylcyclopenta-1,3-dien-1-yl)ethan-1-one (6) via an acid-catalyzed aldol condensation. This chemistry could be relevant for environments characterized by high sulfuric acid concentrations, for example, during the transport of organic compounds from the lower to the upper atmosphere by fast updrafts.

2.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34349018

RESUMO

Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.


Assuntos
Reagentes de Ligações Cruzadas/química , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Chaperoninas/análise , Chaperoninas/química , Chaperoninas/metabolismo , Química Click/métodos , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/química , Complexos Multiproteicos/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Ubiquitina/metabolismo
3.
Anal Chem ; 95(4): 2532-2539, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36652389

RESUMO

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.


Assuntos
Cisteína , Proteoma , Humanos , Proteoma/química , Lisina , Células HEK293 , Peptídeos/química , Espectrometria de Massas/métodos , Sulfóxidos/química , Reagentes de Ligações Cruzadas/química
4.
Mol Cell Proteomics ; 20: 100084, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33915260

RESUMO

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.


Assuntos
Reagentes de Ligações Cruzadas/química , Diazometano/análogos & derivados , Diazometano/química , Cromatografia Líquida , Proteínas Fúngicas/química , Espectrometria de Massas/métodos , Processos Fotoquímicos , Complexo de Endopeptidases do Proteassoma/química , Saccharomyces cerevisiae , Soroalbumina Bovina
5.
Proc Natl Acad Sci U S A ; 117(8): 4088-4098, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32034103

RESUMO

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1-8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN-CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Espectrometria de Massas/métodos , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
6.
Anal Chem ; 94(10): 4236-4242, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35235311

RESUMO

Cross-linking mass spectrometry (XL-MS) is an emergent technology for studying protein-protein interactions (PPIs) and elucidating architectures of protein complexes. The development of various MS-cleavable cross-linkers has facilitated the identification of cross-linked peptides, enabling XL-MS studies at the systems level. However, the scope and depth of cellular networks revealed by current XL-MS technologies remain limited. Due to the inherently broad dynamic range and complexity of proteomes, interference from highly abundant proteins impedes the identification of low-abundance cross-linked peptides in complex samples. Thus, peptide enrichment prior to MS analysis is necessary to enhance cross-link identification for proteome-wide studies. Although chromatographic techniques including size exclusion (SEC) and strong cation exchange (SCX) have been successful in isolating cross-linked peptides, new fractionation methods are still needed to further improve the depth of PPI mapping. Here, we present a two-dimensional (2D) separation strategy by integrating peptide SEC with tip-based high pH reverse-phase (HpHt) fractionation to expand the coverage of proteome-wide XL-MS analyses. Combined with the MS-cleavable cross-linker DSSO, we have successfully mapped in vitro PPIs from HEK293 cell lysates with improved identification of cross-linked peptides compared to existing approaches. The method developed here is effective and can be generalized for cross-linking studies of complex samples.


Assuntos
Espectrometria de Massas , Peptídeos , Proteoma , Fracionamento Químico/métodos , Reagentes de Ligações Cruzadas/química , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Peptídeos/química
7.
Mol Cell Proteomics ; 18(5): 954-967, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723094

RESUMO

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.


Assuntos
Reagentes de Ligações Cruzadas/química , Peróxido de Hidrogênio/toxicidade , Espectrometria de Massas/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Reprodutibilidade dos Testes
8.
Anal Chem ; 92(8): 6026-6033, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32202417

RESUMO

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.


Assuntos
Reagentes de Ligações Cruzadas/síntese química , Safrol/análogos & derivados , Reagentes de Ligações Cruzadas/química , Espectrometria de Massas , Estrutura Molecular , Safrol/química
9.
J Org Chem ; 85(16): 10750-10759, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32806106

RESUMO

The competing enantioselective conversion (CEC) method is a quick and reliable means to determine absolute configuration. Previously, Bode's chiral acylated hydroxamic acids were used to determine the stereochemistry of primary amines, as well as cyclic and acyclic secondary amines. The enantioselective acylation has been evaluated for 4-, 5-, and 6-membered cyclic secondary amines, including medicinally relevant compounds. The limitations of the method were studied through computational analysis and experimental results. Piperidines with substituents at the 2-position did not behave well unless the axial conformer was energetically accessible, which is consistent with the transition state geometries proposed by Bode and Kozlowski. Control experiments were performed to investigate the cause of degrading selectivity under the CEC reaction conditions. The present study expands the scope of the CEC method for secondary amines and provides a better understanding of the reaction profile.

10.
J Am Chem Soc ; 141(34): 13295-13300, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31408328

RESUMO

Illisimonin A was isolated from Illicium simonsii and has a previously unreported tricyclic carbon framework. It displayed neuroprotective effects against oxygen-glucose deprivation-induced cell injury in SH-SY5Y cells. It incorporates a highly strained trans-pentalene ring system. We report the first synthesis of (±)-illisimonin A. Notable steps in the route include a 1,3-dioxa-2-silacyclohexene templated Diels-Alder cycloaddition and type-3 semipinacol rearrangement to generate the trans-pentalene. The final step is an iron-catalyzed C-H oxidation. The synthetic route is robust, with 94 mg of racemic material prepared in a single pass. Resolving an intermediate enabled the synthesis of natural (-)-illisimonin A. The absolute configuration of (-)-illisimonin A was revised to 1S,4S,5S,6S,7R,9R,10R based on the X-ray structure of a heavy-atom analogue.


Assuntos
Illicium/química , Fármacos Neuroprotetores/química , Sesquiterpenos/química , Catálise , Cristalografia por Raios X , Reação de Cicloadição , Frutas/química , Modelos Moleculares , Fármacos Neuroprotetores/síntese química , Sesquiterpenos/síntese química , Estereoisomerismo
11.
Mol Cell Proteomics ; 16(5): 840-854, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28292943

RESUMO

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
12.
Angew Chem Int Ed Engl ; 58(45): 16193-16197, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31491044

RESUMO

(-)-Himeradine A is a complex lycopodium alkaloid with seven rings and ten stereogenic centers that shows anticancer activity against lymphoma L1210 cells. A total synthesis has been developed that builds off prior work on (+)-fastigiatine. A 2,4,6-trisubstitited piperidine ring forms the core of the quinolizidine segment, and was prepared by diastereoselective reduction of a pyridine and classic resolution of an intermediate. The remaining secondary amine was introduced with a catalyst-controlled Overman rearrangement. The piperidine segment was coupled in a B-alkyl Suzuki reaction with a bicyclic bromoenone, which was a key intermediate for the synthesis of (+)-fastigiatine. The final transformation featured a transannular Mannich reaction and cyclization to complete the quinolizidine. Five bonds and four new rings were generated in this one-pot procedure. (-)-Himeradine A was prepared in 17 steps in the longest linear sequence.


Assuntos
Piperidinas/química , Piridinas/química , Quinolizinas/síntese química , Catálise , Ciclização , Estrutura Molecular , Quinolizinas/química , Estereoisomerismo
13.
J Biol Chem ; 292(39): 16310-16320, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28821611

RESUMO

Oxidative stress has been implicated in multiple human neurological and other disorders. Proteasomes are multi-subunit proteases critical for the removal of oxidatively damaged proteins. To understand stress-associated human pathologies, it is important to uncover the molecular events underlying the regulation of proteasomes upon oxidative stress. To this end, we investigated H2O2 stress-induced molecular changes of the human 26S proteasome and determined that stress-induced 26S proteasome disassembly is conserved from yeast to human. Moreover, we developed and employed a new proteomic approach, XAP (in vivo cross-linking-assisted affinity purification), coupled with stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative MS, to capture and quantify several weakly bound proteasome-interacting proteins and examine their roles in stress-mediated proteasomal remodeling. Our results indicate that the adapter protein Ecm29 is the main proteasome-interacting protein responsible for stress-triggered remodeling of the 26S proteasome in human cells. Importantly, using a disuccinimidyl sulfoxide-based cross-linking MS platform, we mapped the interactions of Ecm29 within itself and with proteasome subunits and determined the architecture of the Ecm29-proteasome complex with integrative structure modeling. These results enabled us to propose a structural model in which Ecm29 intrudes on the interaction between the 20S core particle and the 19S regulatory particle in the 26S proteasome, disrupting the proteasome structure in response to oxidative stress.


Assuntos
Modelos Moleculares , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Marcadores de Afinidade , Reagentes de Ligações Cruzadas/farmacologia , Células HEK293 , Humanos , Marcação por Isótopo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/metabolismo
14.
Anal Chem ; 90(12): 7600-7607, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29792801

RESUMO

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. Although such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse, likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS n). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine-reactive cross-linker, bismaleimide sulfoxide (BMSO). We demonstrate that BMSO-cross-linked peptides display the same characteristic fragmentation pattern during collision-induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS n. Additionally, we show that BMSO can complement amine- and acidic-residue-reactive reagents for mapping protein-interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.


Assuntos
Reagentes de Ligações Cruzadas/química , Cisteína/química , Mapeamento de Interação de Proteínas , Soroalbumina Bovina/química , Sulfóxidos/química , Animais , Bovinos , Estrutura Molecular , Ligação Proteica , Sulfóxidos/síntese química , Espectrometria de Massas em Tandem
15.
J Org Chem ; 83(5): 2504-2515, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29424546

RESUMO

Birman's HBTM catalyst is effective for the enantioselective acylation and kinetic resolution of benzylic secondary alcohols. The enantioselective acylation has now been extended to secondary alcohols bearing electron-withdrawing groups such as halides and other heteroatoms. The level of selectivity is modest to good and is sufficient for determining configuration using the competing enantioselective conversion method. A mathematical analysis identifies conditions for achieving maximum differences in conversion and, consequently, assigning configuration with greater confidence. The new method is effective for halohydrins and secondary-tertiary 1,2-diols and was used to confirm the configuration of two inoterpene natural products.

16.
J Org Chem ; 83(16): 8914-8925, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29943989

RESUMO

(+)-Fastigiatine is a complex alkaloid isolated from the alpine club moss Lycopodium fastigatum, most commonly found in New Zealand. It has been the subject of two successful synthetic campaigns. A second-generation route toward fastigiatine was developed to resolve two problematic steps from our initial synthesis. Selective reduction and protection of the C13 ketone improved the yield and reliability of the dibromocarbene ring expansion step. In the prior synthesis, cuprate addition to the C10 enone generated a 1:1 mixture of isomers in an advanced intermediate. Protection of the C13 alcohol with a large silyl group changed the conformational preference of the enone and led to a more selective conjugate addition to produce the desired ß-epimer at C10. MacMillan's decarboxylative photoredox addition method proved to be more practical than the prior aminomethyl cuprate addition chemistry. The second-generation synthesis is longer than the original but improves the selectivity and reproducibility of the overall route.


Assuntos
Alcaloides/química , Alcaloides/síntese química , Técnicas de Química Sintética , Ciclização , Modelos Moleculares , Conformação Molecular
17.
Anal Chem ; 88(20): 10301-10308, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27626298

RESUMO

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.


Assuntos
Reagentes de Ligações Cruzadas/química , Proteínas/química , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Citocromos c/química , Citocromos c/metabolismo , Marcação por Isótopo , Peptídeos/análise , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Sulfóxidos/química
18.
Anal Chem ; 88(16): 8315-22, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27417384

RESUMO

Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein-protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS(n)). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS(n). Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries.


Assuntos
Reagentes de Ligações Cruzadas/química , Peptídeos/química , Safrol/análogos & derivados , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cavalos , Mioglobina/química , Mioglobina/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Safrol/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo
19.
J Org Chem ; 81(22): 10707-10714, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27786473

RESUMO

Several procedures were evaluated for the preparation of lithium 4,4'-di-tert-butylbiphenylide (LiDBB, Freeman's reagent) from lithium metal and 4,4'-di-tert-butylbiphenyl (DBB) in THF. Solutions with nominal concentration of 0.4 and 1.0 M were formed. The stability of LiDBB solutions was evaluated over time, and the gradual uptake of lithium metal was observed. At 0 °C the LiDBB solutions were stable for over a week in THF. At 20 °C the LiDBB solution underwent various decomposition pathways, which led to uptake of more lithium metal and the accumulation of side products. These decomposition pathways were studied, and the importance of ethene in the destruction of THF by LiDBB was observed. On a practical note, LiDBB solutions in THF were stable and effective for over a week at 0 °C or for more than 37 weeks when stored under argon at -25 °C. These observations will extend the utility of LiDBB as a reagent in organic synthesis.

20.
J Org Chem ; 81(21): 10376-10382, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27579766

RESUMO

Pyridine rings are common structural motifs found in a number of biologically active compounds, including some top-selling pharmaceuticals. We have developed a new approach to access substituted pyridines. The method aims to provide a reliable synthesis of a diverse range of substituted pyridines through a three-step procedure. Readily available enones are first converted into 1,5-dicarbonyls through a two-step Hosomi-Sakurai allylation/oxidative cleavage sequence, which is followed by subsequent cyclization to the corresponding pyridine using hydroxylamine hydrochloride. A variety of substituted pyridines have been synthesized using this method.

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