RESUMO
Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other components in the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data.
Assuntos
Algoritmos , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteômica/estatística & dados numéricos , Espectrometria de Massas em Tandem/normas , Calibragem , Humanos , Proteômica/métodos , Razão Sinal-Ruído , Estudos de Validação como AssuntoRESUMO
Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Córtex Cerebral/citologia , Estradiol/farmacologia , Neurônios/fisiologia , Doença de Alzheimer , Animais , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Feto , Humanos , Camundongos , Neuroblastoma , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , Células Tumorais CultivadasRESUMO
Hemoglobin is an important nutrient source for intraerythrocytic malaria organisms. Its catabolism occurs in an acidic digestive vacuole. Our previous studies suggested that an aspartic protease plays a key role in the degradative process. We have now isolated this enzyme and defined its role in the hemoglobinolytic pathway. Laser desorption mass spectrometry was used to analyze the proteolytic action of the purified protease. The enzyme has a remarkably stringent specificity towards native hemoglobin, making a single cleavage between alpha 33Phe and 34Leu. This scission is in the hemoglobin hinge region, unraveling the molecule and exposing other sites for proteolysis. The protease is inhibited by pepstatin and has NH2-terminal homology to mammalian aspartic proteases. Isolated digestive vacuoles make a pepstatin-inhibitable cleavage identical to that of the purified enzyme. The pivotal role of this aspartic hemoglobinase in initiating hemoglobin degradation in the malaria parasite digestive vacuoles is demonstrated.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Hemoglobinas/metabolismo , Malária/sangue , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/isolamento & purificação , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Especificidade por SubstratoRESUMO
During feeding, infected mosquitos inject malaria sporozoites into the host circulation. Within minutes, the parasites are found in the liver where they initiate the first stage of malaria infection. All species of malaria sporozoites are uniformly covered by the circumsporozoite protein (CS), which contains a conserved COOH-terminal sequence called region II-plus. We have previously shown that region II-plus is the parasite's hepatocyte-binding ligand and that this ligand binds to heparan sulfate proteoglycans (HSPGs) on the hepatocyte membrane. Using a series of substituted region II-plus peptides, we show here that the downstream basic amino acids as well as the interdispersed hydrophobic residues are required for binding of CS to hepatocyte HSPGs. We also show that this positively charged stretch of amino acids must be aggregated in order to bind to the receptor. On the basis of this information, we have synthesized a multiple antigen peptide that mimics the hepatocyte-binding ligand. This construct inhibits both CS binding to HepG2 cells in vitro as well as CS clearance in mice.
Assuntos
Fígado/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Apicomplexa/patogenicidade , Feminino , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Proteoglicanas/metabolismo , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.
Assuntos
Ativação Linfocitária , Linfócitos T/imunologia , Vitamina A/farmacologia , Linfócitos B/imunologia , Proteínas Sanguíneas/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Humanos , Cinética , Lipídeos/sangue , Lipídeos/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae , Sítios de Ligação/fisiologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Desoxiglucose/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde , Guanosina Trifosfato/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Luminescentes/genética , Proteínas Nucleares/genética , Porinas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces , Azida Sódica/farmacologia , Staphylococcus aureus/genética , beta Carioferinas , Proteína ran de Ligação ao GTP/metabolismoRESUMO
An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
Assuntos
Proteínas Fúngicas/análise , Proteínas de Membrana/análise , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Proteínas Nucleares/análise , Saccharomyces cerevisiae/ultraestrutura , Transporte Biológico , Proteínas de Transporte/análise , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Imunofluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Imunoeletrônica , Modelos Biológicos , Peso Molecular , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.
Assuntos
Actinas/metabolismo , Anemia Falciforme/metabolismo , Proteínas do Citoesqueleto , Eritrócitos Anormais/metabolismo , Neuropeptídeos , Processamento de Proteína Pós-Traducional , Actinas/química , Sequência de Aminoácidos , Anemia Falciforme/patologia , Simulação por Computador , Membrana Eritrocítica/metabolismo , Eritrócitos Anormais/patologia , Humanos , Substâncias Macromoleculares , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Análise de Sequência , Espectrina/metabolismoRESUMO
Two new technologies have made the study of proteins by mass spectrometry straight-forward. Proteins with molecular masses of up to more than 100 kilodaltons can be analyzed at picomole sensitivities to give simple mass spectra corresponding to the intact molecule. This development has allowed unprecedented accuracy in the determination of the molecular weights of proteins. A number of "case studies" are used to present the revolutionary impact that these powerful new ways of looking at proteins are having on biological research.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Genes , Glicoproteínas/química , Humanos , Lipoproteínas LDL/química , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/químicaRESUMO
Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual low molecular weight cofactor for activity. The cofactor was purified from the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-spermidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis and leishmaniasis.
Assuntos
Coenzimas/isolamento & purificação , Crithidia/enzimologia , Glutationa Redutase/metabolismo , Glutationa/análogos & derivados , Leishmania/enzimologia , Espermidina/análogos & derivados , Trypanosoma/enzimologia , Animais , Fenômenos Químicos , Química , Coenzimas/análise , Coenzimas/metabolismo , Glutationa/análise , Glutationa/isolamento & purificação , Glutationa/metabolismo , Oxirredução , Espermidina/análise , Espermidina/isolamento & purificação , Espermidina/metabolismo , Terminologia como Assunto , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologiaRESUMO
A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.
Assuntos
Sequência de Aminoácidos , Fibrinopeptídeo B/química , Espectrometria de Massas , Análise de Sequência/métodos , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels. This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.
Assuntos
Proteínas de Bactérias , Canais de Potássio/química , Canais de Potássio/metabolismo , Conformação Proteica , Venenos de Escorpião/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Charibdotoxina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Ensaio Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Superfamília Shaker de Canais de Potássio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptomyces/químicaRESUMO
The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.
Assuntos
Proteínas de Bactérias , Canais de Potássio/química , Canais de Potássio/metabolismo , Potássio/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Césio/metabolismo , Cristalização , Cristalografia por Raios X , Análise de Fourier , Ligação de Hidrogênio , Bicamadas Lipídicas , Modelos Moleculares , Dados de Sequência Molecular , Bloqueadores dos Canais de Potássio , Estrutura Secundária de Proteína , Rubídio/metabolismo , Venenos de Escorpião/metabolismo , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Eletricidade Estática , Streptomyces/química , Tetraetilamônio/metabolismo , Tetraetilamônio/farmacologia , ÁguaRESUMO
The gene product of the ob locus is important in the regulation of body weight. The ob product was shown to be present as a 16-kilodalton protein in mouse and human plasma but was undetectable in plasma from C57BL/6J ob/ob mice. Plasma levels of this protein were increased in diabetic (db) mice, a mutant thought to be resistant to the effects of ob. Daily intraperitoneal injections of either mouse or human recombinant OB protein reduced the body weight of ob/ob mice by 30 percent after 2 weeks of treatment with no apparent toxicity but had no effect on db/db mice. The protein reduced food intake and increased energy expenditure in ob/ob mice. Injections of wild-type mice twice daily with the mouse protein resulted in a sustained 12 percent weight loss, decreased food intake, and a reduction of body fat from 12.2 to 0.7 percent. These data suggest that the OB protein serves an endocrine function to regulate body fat stores.
Assuntos
Obesidade/fisiopatologia , Proteínas/farmacologia , Redução de Peso/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Animais , Glicemia/análise , Composição Corporal/efeitos dos fármacos , Diabetes Mellitus/sangue , Diabetes Mellitus/fisiopatologia , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Leptina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/sangue , Obesidade/genética , Proteínas/análise , Proteínas/genética , Proteínas/fisiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologiaRESUMO
The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 A resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ (here referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+ activated K+ channels.
Assuntos
Escherichia coli/química , Canais de Potássio Cálcio-Ativados , Canais de Potássio/química , Receptor trkA , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Cristalização , Cristalografia por Raios X , Bases de Dados Factuais , Condutividade Elétrica , Feminino , Expressão Gênica , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , XenopusRESUMO
GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.
Assuntos
Cromatina/metabolismo , Dano ao DNA , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA , Transcrição Gênica , Ativação Transcricional , Acetilação , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Raios UltravioletaRESUMO
The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.
Assuntos
Bioquímica/métodos , Proteínas/análise , Proteínas/química , Sequência de Aminoácidos , Animais , Biotinilação , Caseínas/química , Bovinos , Embrião de Galinha , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Modelos Químicos , Dados de Sequência Molecular , Ovalbumina/química , Fosforilação , Fosfosserina/química , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Treonina/químicaRESUMO
The extracellular hemoglobin of the earthworm has four major O2-binding chains, a, b, c and d, together with additional non-heme structural chains that are required for assembly. Although the abc trimer self-associates extensively at least to (abc)10, addition of chain d results in the formation of a discrete 280 kDa complex corresponding to (abcd)4. Thus a primary function of chain d is to cap the abc association and convert an abc trimer that binds O2 with weak cooperativity to a highly cooperative (abcd)4 complex. Amino-acid sequences of the major globin chains a, b, c have been determined previously by peptide and cDNA analysis. However, the peptide sequence reported for the major chain d (Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N. and Walz, D.A. (1987) J. Biol. Chem., 262. 3123-3131), has a calculated molecular mass 134-167 Da higher than masses for components of chain d determined by mass spectrometry (Owrby, D.W., Zhu, H., Schneider, K., Beavis, R.C., Chait, B.T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13539-13547). Reverse-phase HPLC confirms the presence of two distinct polypeptides, d1 and d2, together with d'1, a variant of d1, cDNA derived amino acid sequences have been determined for chains d'1 and d2 by application of the polymerase chain reaction with primers based on the NH2-terminal sequences and oligo-dT. Each of the two cDNA-derived sequences has 140 residues and they differ by 28 substitutions. The data show that the sequence originally reported had been derived from peptides generated from both polypeptides.
Assuntos
Hemoglobinas/química , Oligoquetos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , Globinas/química , Globinas/genética , Globinas/isolamento & purificação , Hemoglobinas/genética , Hemoglobinas/isolamento & purificação , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Oligoquetos/genética , Conformação ProteicaRESUMO
Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Therefore, there is a great need for methods capable of accurately elucidating sites of phosphorylation. Although full characterization of phosphoproteins remains a formidable analytical challenge, mass spectrometry has emerged as an increasingly viable tool for this task. This review summarizes the methodologies currently available for the analysis of phosphoproteins by mass spectrometry, including enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Fosforilação , Proteínas/análise , Animais , Previsões , Humanos , Peptídeos/química , Proteínas/químicaRESUMO
Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon. It has been proposed that PCNA functions by forming a trimeric complex with a ring-like structure through which DNA is threaded. PCNA from the yeast Saccharomyces cerevisiae has been crystallized in a cubic space group (P2(1)3, a = 121.1 A). Unexpectedly, a mercury derivative of PCNA yields crystals that diffract significantly better than crystals of the unmodified protein (2.4 A and 3.0 A resolution, respectively). Mass spectrometry reveals that the derivative results from the addition of two mercury atoms to the protein. Although crystals of the mercurated protein show evidence of non-isomorphism, the anomalous diffraction signal is strong and phases may be determined by multi-wavelength anomalous diffraction (MAD phasing).