RESUMEN

Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones. The technique was further refined to improve coverage of hydrophilic as well as hydrophobic peptides and subsequently applied to human pancreatic islet cultures derived from normal donors or donors with type 2 diabetes. Interestingly, in addition to the expected islet hormones, we identified alpha-cell-derived bioactive GLP-1, consistent with recent reports of paracrine effects of this hormone on beta-cell function. We also identified many novel peptides derived from neurohormonal precursors and proteins related to the cell secretory system. Taken together, these results suggest the PHASST-MS strategy of focusing on cellular secreted products rather than the total tissue peptidome may improve the probability of discovering novel bioactive peptides and also has the potential to offer important new insights into the secretion and function of known hormones.

Asunto(s)

Diabetes Mellitus Tipo 2/metabolismo , Péptido 1 Similar al Glucagón/análisis , Islotes Pancreáticos/metabolismo , Hormonas Peptídicas/análisis , Proteómica/métodos , Secuencias de Aminoácidos , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Ratones , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Hormonas Peptídicas/química , Hormonas Peptídicas/metabolismo , Técnicas de Cultivo de Tejidos

RESUMEN

To enable the first physiologically relevant peptidomic survey of gastrointestinal tissue, we have developed a primary mouse colonic crypt model enriched for enteroendocrine L-cells. The cells in this model were phenotypically profiled using PCR-based techniques and showed peptide hormone and secretory and processing marker expression at mRNA levels that were increased relative to the parent tissue. Co-localization of glucagon-like peptide-1 and peptide YY, a characteristic feature of L-cells, was demonstrated by double label immunocytochemistry. The L-cells displayed regulated hormone secretion in response to physiological and pharmacological stimuli as measured by immunoassay. Using a high resolution mass spectrometry-based platform, more than 50 endogenous peptides (<16 kDa), including all known major hormones, were identified a priori. The influence of culture conditions on peptide relative abundance and post-translational modification was characterized. The relative abundance of secreted peptides in the presence/absence of the stimulant forskolin was measured by label-free quantification. All peptides exhibiting a statistically significant increase in relative concentration in the culture media were derived from prohormones, consistent with a cAMP-coupled response. The only peptides that exhibited a statistically significant decrease in secretion on forskolin stimulation were derived from annexin A1 and calcyclin. Biophysical interactions between annexin A1 and calcyclin have been reported very recently and may have functional consequences. This work represents the first step in characterizing physiologically relevant peptidomic secretion of gastrointestinally derived primary cells and will aid in elucidating new endocrine function.

Asunto(s)

Colon/citología , Células Enteroendocrinas/citología , Hormonas Gastrointestinales/metabolismo , Mucosa Intestinal/citología , Péptidos/análisis , Algoritmos , Secuencia de Aminoácidos , Animales , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Colon/metabolismo , Medios de Cultivo/química , Células Enteroendocrinas/metabolismo , Hormonas Gastrointestinales/química , Mucosa Intestinal/metabolismo , Metaboloma , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Manejo de Especímenes/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

An unusual sulfotyrosine-, phosphoserine-containing motif was mapped on a differentially post-translationally modified 60 residue antimicrobial neuroendocrine peptide called chrombacin. The study was performed by high resolution FT MS using complementary fragmentation techniques. The peptide was analyzed at low levels directly from cell culture media in contrast to previous reports that required extensive purification and proteolytic digestion. The sulfation site was not previously described nor predicted by informatic analysis of the peptide's precursor sequence.

Asunto(s)

Cromogranina B/química , Neuropéptidos/química , Fosfopéptidos/química , Ésteres del Ácido Sulfúrico/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular Tumoral , Cromogranina B/metabolismo , Medios de Cultivo Condicionados/química , Perros , Humanos , Ratones , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Fosfopéptidos/metabolismo , Fosforilación , Fosfoserina/química , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ésteres del Ácido Sulfúrico/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Tirosina/genética

RESUMEN

A novel approach is presented for the simultaneous identification and relative quantification of secreted peptides, particularly those that have been historically difficult to analyze in a concerted manner. Peptides exceeding 60 residues with various degrees of post-translational modification were identified on a liquid chromatographic time scale. The approach demonstrates high efficiency pattern-based recognition analysis of complex neuroendocrine peptide sets and enables rapid identification of biomarkers from biological material.

Asunto(s)

Biomarcadores de Tumor/análisis , Evaluación Preclínica de Medicamentos/métodos , Hormonas/química , Insulinoma/química , Neoplasias Pancreáticas/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Colforsina/farmacología , Simulación por Computador , Medios de Cultivo Condicionados/química , Insulinoma/patología , Datos de Secuencia Molecular , Neoplasias Pancreáticas/patología , Proteómica/métodos , Ratas

RESUMEN

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both beta4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.

Asunto(s)

Citoplasma/metabolismo , Estrés Oxidativo , Proteína de la Poliposis Adenomatosa del Colon/química , Animales , Biotina/química , División Celular , Citosol/metabolismo , Disulfuros/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Epítopos , Vectores Genéticos , Glutatión/química , Proteínas HSP70 de Choque Térmico/química , Humanos , Peróxido de Hidrógeno/química , Immunoblotting , Yodoacetamida/química , Espectrometría de Masas , Ratones , Neuronas/metabolismo , Nucleósido-Difosfato Quinasa/química , Oxidación-Reducción , Oxígeno/metabolismo , Peroxidasas/química , Peroxirredoxinas , Pruebas de Precipitina , Especies Reactivas de Oxígeno , Ribonucleótido Reductasas/química , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Reductasa de Tiorredoxina-Disulfuro/química

RESUMEN

We describe a simple, rapid method for protein complex purification in planta. Using a biotin peptide as an affinity tag with TATA-box binding protein (TBP), 86 unique proteins present in the purified complex were identified by tandem mass spectrometry. We identified proteins known to be associated with TBP, and many other proteins involved in pre-mRNA processing and chromatin remodeling. The identification of these novel protein-protein associations will upon further investigations provide new insights into the mechanisms of mRNA transcription and pre-mRNA processing.

Asunto(s)

Oryza/metabolismo , Proteínas/aislamiento & purificación , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Biotina/química , Western Blotting , Cromatografía Líquida de Alta Presión , Secuencia Conservada , Bases de Datos Factuales , Espectrometría de Masas , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Tinción con Nitrato de Plata , Factores de Transcripción/genética , Zea mays/genética

RESUMEN

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.

Asunto(s)

Lectina de Unión a Manosa/química , Oryza/metabolismo , Proteoma , Unión Competitiva , Carbohidratos/química , Cromatografía Líquida de Alta Presión , Bases de Datos como Asunto , Genoma de Planta , Lectinas , Ligandos , Manosa/química , Lectina de Unión a Manosa/aislamiento & purificación , Espectrometría de Masas

RESUMEN

We describe the identification of a previously uncharacterized plant virus that is capable of infecting Nicotiana spp. and Arabidopsis thaliana. Protein extracts were first prepared from leaf tissue of uninfected tobacco plants, and the proteins were visualized with two-dimensional electrophoresis (2-DE). Matching gels were then run using protein extracts of a tobacco plant infected with tobacco mosaic virus (TMV). After visual comparison, the proteins spots that were differentially expressed in infected plant tissues were cut from the gels and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Tandem mass spectrometry data of individual peptides was searched with SEQUEST. Using this approach we demonstrated a successful proof-of-concept experiment by identifying TMV proteins present in the total protein extract. The same procedure was then applied to tobacco plants infected with a laboratory viral isolate of unknown identity. Several of the differentially expressed protein spots were identified as proteins of potato virus X (PVX), thus successfully identifying the causative agent of the uncharacterized viral infection. We believe this demonstrates that HPLC-MS/MS can be used to successfully characterize unknown viruses in infected plants.

Asunto(s)

Nicotiana/virología , Virus de Plantas/química , Virus de Plantas/clasificación , Proteoma/análisis , Proteómica/métodos , Proteínas Virales/análisis , Secuencia de Aminoácidos , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Datos de Secuencia Molecular , Hojas de la Planta/química , Hojas de la Planta/virología , Virus de Plantas/aislamiento & purificación , Potexvirus/química , Potexvirus/clasificación , Potexvirus/aislamiento & purificación , Proteoma/química , Proteínas Virales/química

Asunto(s)

Proteómica , Electroforesis en Gel Bidimensional , Espectrometría de Masas

RESUMEN

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

Asunto(s)

Espectrometría de Masas/métodos , Proteoma , Triticum/química , Cromatografía Líquida de Alta Presión , Bases de Datos como Asunto , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Proteínas de Plantas/química , Triticum/metabolismo

RESUMEN

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Asunto(s)

Oryza/metabolismo , Proteoma , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido