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1.
J Proteome Res ; 23(4): 1144-1149, 2024 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-38412507

RESUMEN

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease. Knowledge of a patient's apoE genotype has high diagnostic value. A recent study has introduced an LC-MRM-MS-based proteomic approach for apoE isoform genotyping using stable isotope-labeled peptide internal standards (SIS). Here, our goal was to develop a simplified LC-MRM-MS assay for identifying apoE genotypes in plasma samples, eliminating the need for the use of SIS peptides. To determine the apoE genotypes, we monitored the chromatographic peak area ratios of isoform-specific peptides relative to a peptide that is common to all apoE isoforms. The assay results correlated well with the standard TaqMan allelic discrimination assay, and we observed a concordance between the two methods for all but three out of 172 samples. DNA sequencing of these three samples has confirmed that the results of the LC-MRM-MS method were correct. Thus, our simplified UPLC-MRM-MS assay is a feasible and reliable method for identifying apoE genotypes without using SIS internal standard peptides. The approach can be seamlessly incorporated into existing quantitative proteomics assays and kits, providing additional valuable apoE genotype information. The principle of using signal ratios of the protein isoform-specific peptides to the peptide common for all of the protein isoforms has the potential to be used for protein isoform determination in general.


Asunto(s)
Enfermedad de Alzheimer , Proteómica , Humanos , Apolipoproteínas E/genética , Genotipo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Alelos , Isoformas de Proteínas/genética , Péptidos/genética
2.
Chem Rev ; 122(8): 7488-7499, 2022 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-34968047

RESUMEN

The advent of soft-ionization mass spectrometry for biomolecules has opened up new possibilities for the structural analysis of proteins. Combining protein chemistry methods with modern mass spectrometry has led to the emergence of the distinct field of structural proteomics. Multiple protein chemistry approaches, such as surface modification, limited proteolysis, hydrogen-deuterium exchange, and cross-linking, provide diverse and often orthogonal structural information on the protein systems studied. Combining experimental data from these various structural proteomics techniques provides a more comprehensive examination of the protein structure and increases confidence in the ultimate findings. Here, we review various types of experimental data from structural proteomics approaches with an emphasis on the use of multiple complementary mass spectrometric approaches to provide experimental constraints for the solving of protein structures.


Asunto(s)
Proteínas , Proteómica , Espectrometría de Masas/métodos , Proteínas/química , Proteolisis , Proteómica/métodos
3.
J Proteome Res ; 22(9): 3096-3102, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37526474

RESUMEN

Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the structure and the location of protein interaction sites. Here, we have characterized a monoclonal antibody epitope for a protein antigen by a combination of differential photoreactive surface modification (SM), cross-linking (CL), differential hydrogen-deuterium exchange (HDX), and epitope extraction/excision. We found that experimental data from different approaches agree with each other in determining the epitope of the monoclonal antibody on the protein antigens using the HIV-1 p24-mAb E complex as an illustrative example. A combination of these multiple structural proteomics approaches results in a detailed picture of the interaction of the proteins and increases confidence in the determination of the final structure of the protein interaction interface. Data are available via ProteomeXchange with identifier PXD040902.


Asunto(s)
Anticuerpos Monoclonales , Proteómica , Epítopos/química , Anticuerpos Monoclonales/química , Mapeo Epitopo/métodos , Espectrometría de Masas/métodos
4.
Anal Bioanal Chem ; 415(22): 5261-5267, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37468754

RESUMEN

Amino acid analysis (AAA) can be used for absolute quantitation of standard peptides after acid hydrolysis using 6 M HCl. Obtained individual amino acids can then be quantified by liquid chromatography-mass spectrometry (LC-MS). Achieving baseline separation of non-derivatized amino acids is challenging when reversed-phase (RP) chromatography is used. Several derivatization methods are commonly utilized to address this issue; however, derivatization has several drawbacks, such as derivative instability and lack of reproducibility. Currently, separation of non-derivatized amino acids is typically done using HILIC, but HILIC has problems of poor reproducibility and long column equilibration times. We developed a method to quantify non-derivatized amino acids, including methionine and cysteine, from peptide hydrolysates by RP-LC-MS without special pre-treatment of the samples. Samples were spiked with certified isotopically labeled (13C- and/or 15N-) amino acids as internal standards. The amino acids released from acid hydrolysis were then analyzed by RP-UPLC-MRM-MS and quantified using the analyte/internal standard chromatographic peak area ratios. Peptide quantitation was based on the sum of the individual amino acid concentrations from the known peptide sequences. The resulting method did not require derivatization, used standard C18-based reversed-phase liquid chromatography, did not require external calibration, was robust, and was able to quantify all 17 amino acids for which we had internal standards, including the sulfur-containing amino acids, cysteine and methionine, in their respective oxidized forms. This simple and robust method enabled the absolute quantitation of standard peptides using only acid hydrolysis and a standard RP-UPLC-MRM-MS setup.


Asunto(s)
Aminoácidos , Cromatografía de Fase Inversa , Aminoácidos/análisis , Cisteína , Reproducibilidad de los Resultados , Espectrometría de Masas/métodos , Péptidos , Aminas , Metionina , Cromatografía Líquida de Alta Presión/métodos
5.
Mol Cell Proteomics ; 19(4): 624-639, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32051233

RESUMEN

An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Marcaje Isotópico , Espectrometría de Masas , Orgánulos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Biotina/análogos & derivados , Fraccionamiento Químico , Mitocondrias/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/metabolismo , Succinimidas
6.
Proteomics ; 21(21-22): e2000298, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34482645

RESUMEN

The conversion of the native monomeric cellular prion protein (PrPC ) into an aggregated pathological ß-oligomeric form (PrPß ) and an infectious form (PrPSc ) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational changes involved in the conversion are still unknown. We applied mass spectrometry combined with chemical crosslinking, hydrogen/deuterium exchange, limited proteolysis, and surface modification for the differential characterization of the native and the urea+acid-converted prion ß-oligomer structures to obtain insights into the mechanisms of conversion and aggregation. For the determination of the structure of the monomer and the dimer unit of the ß-oligomer, we applied a recently-developed approach for de novo protein structure determination which is based on the incorporation of zero-length and short-distance crosslinking data as intra- and inter-protein constraints in discrete molecular dynamics simulations (CL-DMD). Based on all of the structural-proteomics experimental data and the computationally predicted structures of the monomer units, we propose the potential mode of assembly of the ß-oligomer. The proposed ß-oligomer assembly provides a clue on the ß-sheet nucleation site, and how template-based conversion of the native prion molecule occurs, growth of the prion aggregates, and maturation into fibrils may occur.


Asunto(s)
Priones , Espectrometría de Masas , Simulación de Dinámica Molecular , Conformación Proteica , Conformación Proteica en Lámina beta , Pliegue de Proteína , Proteómica
7.
J Struct Biol ; 213(2): 107733, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33819634

RESUMEN

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.


Asunto(s)
Azúcares de Nucleósido Difosfato/biosíntesis , Nucleotidiltransferasas/química , Oxidorreductasas/química , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Pared Celular/metabolismo , Cristalografía por Rayos X , Espectrometría de Masas/métodos , Modelos Moleculares , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Oxidorreductasas/metabolismo , Pentosafosfatos/metabolismo , Multimerización de Proteína , Ribulosafosfatos/metabolismo , Staphylococcus aureus/citología , Staphylococcus aureus/enzimología
8.
PLoS Comput Biol ; 15(3): e1006859, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30917118

RESUMEN

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently-developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD) for the determination of conformational ensemble of the intrinsically disordered protein α-synuclein in the solution. The predicted structures were in agreement with hydrogen-deuterium exchange, circular dichroism, surface modification, and long-distance crosslinking data. We found that α-synuclein is present in solution as an ensemble of rather compact globular conformations with distinct topology and inter-residue contacts, which is well-represented by movements of the large loops and formation of few transient secondary structure elements. Non-amyloid component and C-terminal regions were consistently found to contain ß-structure elements and hairpins.


Asunto(s)
Simulación de Dinámica Molecular , alfa-Sinucleína/química , alfa-Sinucleína/ultraestructura , Humanos , Conformación Proteica , Proteómica , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructura
9.
Nucleic Acids Res ; 45(20): 11989-12004, 2017 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-29036638

RESUMEN

Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.


Asunto(s)
Conformación de Ácido Nucleico , Dominios Proteicos , Estructura Secundaria de Proteína , ARN Bicatenario/química , Proteínas de Unión a Tacrolimus/química , Secuencia de Bases , Western Blotting , Dominio Catalítico , Línea Celular Tumoral , Células HEK293 , Humanos , Microscopía Confocal , Modelos Moleculares , Unión Proteica , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo
10.
Anal Chem ; 90(5): 3079-3082, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29336549

RESUMEN

Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

11.
RNA ; 20(7): 1014-22, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24840943

RESUMEN

Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit.


Asunto(s)
Fosfoproteínas/metabolismo , Precursores de Proteínas/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Unión Proteica , Precursores del ARN/metabolismo , ARN Ribosómico 28S/metabolismo , Proteínas de Unión a Tacrolimus/genética , Nucleolina
12.
Methods ; 89: 74-8, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-25752848

RESUMEN

Disulfide bonds are valuable constraints in protein structure modeling. The Cys-Cys disulfide bond undergoes specific fragmentation under CID and, therefore, can be considered as a CID-cleavable crosslink. We have recently reported on the benefits of using non-specific digestion with proteinase K for inter-peptide crosslink determination. Here, we describe an updated application of our CID-cleavable crosslink analysis software and our crosslinking analysis with non-specific digestion methodology for the robust and comprehensive determination of disulfide bonds in proteins, using Orbitrap LC/ESI-MS/MS data.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Cisteína/análisis , Disulfuros/análisis , Endopeptidasa K/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Reactivos de Enlaces Cruzados/metabolismo , Cisteína/metabolismo , Disulfuros/metabolismo , Endopeptidasa K/metabolismo , Humanos
13.
Nucleic Acids Res ; 41(20): 9266-73, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23939621

RESUMEN

The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine-lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.


Asunto(s)
Complejo Mediador/química , Modelos Moleculares , Complejo Mediador/genética , Complejo Mediador/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química
14.
Proteomics ; 14(17-18): 1987-9, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24895266

RESUMEN

Cross-linking combined with MS involves enzymatic digestion of cross-linked proteins and identifying cross-linked peptides. Assignment of cross-linked peptide masses requires a search of all possible binary combinations of peptides from the cross-linked proteins' sequences, which becomes impractical with increasing complexity of the protein system and/or if digestion enzyme specificity is relaxed. Here, we describe the application of a fast sorting algorithm to search large sequence databases for cross-linked peptide assignments based on mass. This same algorithm has been used previously for assigning disulfide-bridged peptides (Choi et al., ), but has not previously been applied to cross-linking studies.


Asunto(s)
Bases de Datos de Proteínas , Péptidos/análisis , Péptidos/química , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Algoritmos , Espectrometría de Masas/métodos
15.
J Proteome Res ; 13(2): 527-35, 2014 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-24354799

RESUMEN

SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probability that most of the proteolytic peptides produced will be present in the final digest solution. The resulting peptide mixture is then freed of SDS and concentrated using SCX (strong cation exchange) zip-tips and analyzed by mass spectrometry. For standard protein identification studies and the recovery of noncross-linked peptides, the in-gel procedure outperformed the out-gel procedure, but for cross-linking studies with enrichable cross-linkers (such as CBDPS), the standard out-gel procedure allowed the recoveries of cross-links not recovered via the in-gel method. Labeling experiments showed that, with an enrichable cross-linker, 93% of the cross-links showed better or equal recoveries with the out-gel procedure, as compared to the in-gel procedure. It should be noted that this method is not designed to replace in-gel digestion for most proteomics applications. However, by using the out-gel method, we were able to detect twice as many interprotein CBDPS cross-links from the histone H2A/H2B complex as were found in the in-gel digested sample.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tripsina/metabolismo , Electroforesis en Gel de Poliacrilamida , Transcriptasa Inversa del VIH/metabolismo
16.
J Biol Chem ; 288(18): 12805-17, 2013 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-23511632

RESUMEN

Plasmodium falciparum is the most devastating agent of human malaria. A major contributor to its virulence is a complex lifecycle with multiple parasite forms, each presenting a different repertoire of surface antigens. Importantly, members of the 6-Cys s48/45 family of proteins are found on the surface of P. falciparum in every stage, and several of these antigens have been investigated as vaccine targets. Pf12 is the archetypal member of the 6-Cys protein family, containing just two s48/45 domains, whereas other members have up to 14 of these domains. Pf12 is strongly recognized by immune sera from naturally infected patients. Here we show that Pf12 is highly conserved and under purifying selection. Immunofluorescence data reveals a punctate staining pattern with an apical organization in late schizonts. Together, these data are consistent with an important functional role for Pf12 in parasite-host cell attachment or invasion. To infer the structural and functional diversity between Pf12 and the other 11 6-Cys domain proteins, we solved the 1.90 Å resolution crystal structure of the Pf12 ectodomain. Structural analysis reveals a unique organization between the membrane proximal and membrane distal domains and clear homology with the SRS-domain containing proteins of Toxoplasma gondii. Cross-linking and mass spectrometry confirm the previously identified Pf12-Pf41 heterodimeric complex, and analysis of individual cross-links supports an unexpected antiparallel organization. Collectively, the localization and structure of Pf12 and details of its interaction with Pf41 reveal important insight into the structural and functional properties of this archetypal member of the 6-Cys protein family.


Asunto(s)
Antígenos de Protozoos/química , Plasmodium falciparum/química , Esquizontes/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Humanos , Plasmodium falciparum/genética , Estructura Terciaria de Proteína , Esquizontes/inmunología
17.
Mol Cell Proteomics ; 11(7): M111.013524, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22438564

RESUMEN

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPß). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPß prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPß and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.


Asunto(s)
Endopeptidasa K/metabolismo , Péptidos/análisis , Priones/análisis , Secuencia de Aminoácidos , Animales , Biotina , Cromatografía de Afinidad , Cricetinae , Reactivos de Enlaces Cruzados , Escherichia coli , Mesocricetus , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Priones/química , Priones/genética , Proteolisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
18.
Nucleic Acids Res ; 40(7): 2884-97, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22144686

RESUMEN

Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.


Asunto(s)
Encéfalo/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Nucleosomas/metabolismo , Animales , Núcleo Celular/metabolismo , Cromatina/ultraestructura , ADN/metabolismo , Metilación de ADN , Desoxirribonucleasas , Células HeLa , Histonas/química , Humanos , Neuronas/metabolismo , Neuronas/ultraestructura , Unión Proteica , Procesamiento Proteico-Postraduccional , Ratas
19.
Mol Cell Proteomics ; 10(2): M110.001420, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20622150

RESUMEN

Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.


Asunto(s)
Biotinilación , Reactivos de Enlaces Cruzados/farmacología , Proteómica/métodos , Biotina/análogos & derivados , Biotina/química , Cromatografía/métodos , Reactivos de Enlaces Cruzados/química , Dimerización , Transcriptasa Inversa del VIH/metabolismo , Espectrometría de Masas/métodos , Modelos Químicos , Péptidos/química , Proteínas/química , Proteoma , Succinimidas/química , Espectrometría de Masas en Tándem/métodos
20.
J Clin Invest ; 133(9)2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-37115698

RESUMEN

Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how inflammatory cells affect cardiomyocytes in the remodeling heart. Pathogenic cardiac macrophages induced an IFN response in cardiomyocytes, characterized by upregulation of the ubiquitin-like protein IFN-stimulated gene 15 (ISG15), which posttranslationally modifies its targets through a process termed ISGylation. Cardiac ISG15 is controlled by type I IFN signaling, and ISG15 or ISGylation is upregulated in mice with transverse aortic constriction or infused with angiotensin II; rats with uninephrectomy and DOCA-salt, or pulmonary artery banding; cardiomyocytes exposed to IFNs or CD4+ T cell-conditioned medium; and ventricular tissue of humans with nonischemic cardiomyopathy. By nanoscale liquid chromatography-tandem mass spectrometry, we identified the myofibrillar protein filamin-C as an ISGylation target. ISG15 deficiency preserved cardiac function in mice with transverse aortic constriction and led to improved recovery of mouse hearts ex vivo. Metabolomics revealed that ISG15 regulates cardiac amino acid metabolism, whereas ISG15 deficiency prevented misfolded filamin-C accumulation and induced cardiomyocyte autophagy. In sum, ISG15 upregulation is a feature of pathological ventricular remodeling, and protein ISGylation is an inflammation-induced posttranslational modification that may contribute to heart failure development by altering cardiomyocyte protein turnover.


Asunto(s)
Citocinas , Insuficiencia Cardíaca , Humanos , Ratas , Ratones , Animales , Citocinas/genética , Citocinas/metabolismo , Filaminas , Remodelación Ventricular/genética , Insuficiencia Cardíaca/metabolismo , Inflamación , Ubiquitinas/genética
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