RESUMEN
Serum is the body fluid most often used in biomarker discovery. Albumin, the most abundant serum protein, contributes approximately 50% of the serum protein content, with an additional dozen abundant proteins dominating the rest of the serum proteome. To profile this challenging protein mixture by proteomics, the abundant proteins must be depleted to allow for detection of the low-abundant proteins, the primary biomarker targets. Current serum depletion approaches for proteomics are costly and relatively complex to couple with protein digestion. We demonstrate a simple, affordable serum depletion methodology that, within a few minutes of processing, results in two captured serum fractions - albumin-depleted and albumin-rich - which are digested in situ. We believe our method is a useful addition to the biomarker sample preparation toolbox.
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Proteínas Sanguíneas , Proteoma , Proteómica/métodos , Biomarcadores/sangre , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Cromatografía de Fase Inversa , Humanos , Proteoma/análisis , Proteoma/química , Proteoma/aislamiento & purificaciónRESUMEN
BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. METHODS: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. RESULTS: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. CONCLUSIONS: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathway.
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3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Carcinoma de Células Renales/genética , Citocinas/genética , Quinurenina 3-Monooxigenasa/genética , Quinurenina/genética , Nicotinamida Fosforribosiltransferasa/genética , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Quinurenina/metabolismo , Redes y Vías Metabólicas/genética , Proteómica , Escape del Tumor/genética , Escape del Tumor/inmunologíaRESUMEN
The integration of omics techniques has seen a step change in our understanding of biological systems. However, multiomics has been impaired by mutually exclusive omic separation methods and the destructive nature of the techniques when sample is limited. We describe Simultaneous Trapping (SiTrap), a simple and effective detergent-free method that facilitates direct measurement of the proteome and metabolome in the same sample extract. This "single-pot" multiomics processing is particularly beneficial in cases when sample amounts are limited or are heterogeneous, for example, tissue biopsies. We demonstrate the value of the SiTrap methodology as an essential multiomics tool in a proof-of-principle integrated study of renal cancer tissue biopsy samples. We believe SiTrap has the potential to become an indispensable tool in translational medical research.
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Metabolómica , Proteómica , Metaboloma , ProteomaRESUMEN
Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of Ab-secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. In this study, we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation network analysis (PGCNA), a new approach established by our group, we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGF-ß3. This analysis demonstrates a convergent pattern of differentiation, linking unfolded protein response/endoplasmic reticulum stress to secretory optimization, coordinated with cell cycle exit. TGF-ß3 supports ASC survival while having a limited effect on gene expression including upregulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.
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Células Productoras de Anticuerpos/inmunología , Quimiocina CXCL12/inmunología , Factor de Crecimiento Transformador beta3/inmunología , Supervivencia Celular , Células Cultivadas , Quimiocina CXCL12/genética , Voluntarios Sanos , Humanos , Factor de Crecimiento Transformador beta3/genéticaRESUMEN
Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors.
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Modelos Anatómicos , Recurrencia Local de Neoplasia/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/cirugía , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Células Clonales , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Masculino , Ratones Desnudos , Modelos Estadísticos , Células Madre Neoplásicas/patología , Neprilisina/metabolismo , Fenotipo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Plasma cells (PCs) as effectors of humoral immunity produce Igs to match pathogenic insult. Emerging data suggest more diverse roles exist for PCs as regulators of immune and inflammatory responses via secretion of factors other than Igs. The extent to which such responses are preprogrammed in B-lineage cells or can be induced in PCs by the microenvironment is unknown. In this study, we dissect the impact of IFNs on the regulatory networks of human PCs. We show that core PC programs are unaffected, whereas PCs respond to IFNs with distinctive transcriptional responses. The IFN-stimulated gene 15 (ISG15) system emerges as a major transcriptional output induced in a sustained fashion by IFN-α in PCs and linked both to intracellular conjugation and ISG15 secretion. This leads to the identification of ISG15-secreting plasmablasts/PCs in patients with active systemic lupus erythematosus. Thus, ISG15-secreting PCs represent a distinct proinflammatory PC subset providing an Ig-independent mechanism of PC action in human autoimmunity.
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Autoinmunidad/inmunología , Citocinas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Células Plasmáticas/inmunología , Transcriptoma , Ubiquitinas/metabolismo , Western Blotting , Citocinas/inmunología , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Interferón-alfa/inmunología , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Ubiquitinas/inmunologíaRESUMEN
Recently we introduced the concept of Suspension Trapping (STrap) for bottom-up proteomics sample processing that is based upon SDS-mediated protein extraction, swift detergent removal and rapid reactor-type protein digestion in a quartz depth filter trap. As the depth filter surface is made of silica, it is readily modifiable with various functional groups using the silane coupling chemistries. Thus, during the digest, peptides possessing specific features could be targeted for enrichment by the functionalized depth filter material while non-targeted peptides could be collected as an unbound distinct fraction after the digest. In the example presented here the quartz depth filter surface is functionalized with the pyridyldithiol group therefore enabling reversible in-situ capture of the cysteine-containing peptides generated during the STrap-based digest. The described C-STrap method retains all advantages of the original STrap methodology and provides robust foundation for the conception of the targeted in-situ peptide fractionation in the STrap format for bottom-up proteomics. The presented data support the method's use in qualitative and semi-quantitative proteomics experiments.
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Péptidos , Proteómica/métodos , Cromatografía Liquida/métodos , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas/métodos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Despite recent developments in bottom-up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.
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Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/aislamiento & purificación , Proteínas/análisis , Proteínas/aislamiento & purificación , Proteómica/métodos , Detergentes/química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoprecipitación , Fragmentos de Péptidos/química , Proteínas/química , Dodecil Sulfato de Sodio/químicaRESUMEN
Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs.
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Neoplasias de la Mama/metabolismo , Movimiento Celular , Fibroblastos/metabolismo , MicroARNs/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Técnicas de Cocultivo , Regulación hacia Abajo , Femenino , Fibroblastos/patología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina , Reacción en Cadena de la Polimerasa , Transducción de Señal , Factores de Tiempo , Transfección , Microambiente TumoralRESUMEN
PURPOSE: The aim of this study was to identify and validate novel predictive and/or prognostic serum proteomic biomarkers in patients with epithelial ovarian cancer (EOC) treated as part of the phase III international ICON7 clinical trial. EXPERIMENTAL DESIGN: ICON7 was a phase III international trial in EOC which showed a modest but statistically significant benefit in progression-free survival (PFS) with the addition of bevacizumab to standard chemotherapy. Serum samples from 10 patients who received bevacizumab (five responders and five nonresponders) were analyzed by mass spectrometry to identify candidate biomarkers. Initial validation and exploration by immunoassay was undertaken in an independent cohort of 92 patients, followed by a second independent cohort of 115 patients (taken from across both arms of the trial). RESULTS: Three candidate biomarkers were identified: mesothelin, fms-like tyrosine kinase-4 (FLT4), and α1-acid glycoprotein (AGP). Each showed evidence of independent prognostic potential when adjusting for high-risk status in initial (P < 0.02) and combined (P < 0.01) validation cohorts. In cohort I, individual biomarkers were not predictive of bevacizumab benefit; however, when combined with CA-125, a signature was developed that was predictive of bevacizumab response and discriminated benefit attributable to bevacizumab better than clinical characteristics. The signature showed weaker evidence of predictive ability in validation cohort II, but was still strongly predictive considering all samples (P = 0.001), with an improvement in median PFS of 5.5 months in signature-positive patients in the experimental arm compared with standard arm. CONCLUSIONS: This study shows a discriminatory signature comprising mesothelin, FLT4, AGP, and CA-125 as potentially identifying those patients with EOC more likely to benefit from bevacizumab. These results require validation in further patient cohorts.
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Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias de las Trompas Uterinas/sangre , Neoplasias Ováricas/sangre , Selección de Paciente , Neoplasias Peritoneales/sangre , Adenocarcinoma de Células Claras/sangre , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/mortalidad , Adenocarcinoma Mucinoso/sangre , Adenocarcinoma Mucinoso/tratamiento farmacológico , Adenocarcinoma Mucinoso/mortalidad , Adulto , Anciano , Bevacizumab , Cromatografía Liquida , Estudios de Cohortes , Cistadenocarcinoma Seroso/sangre , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/mortalidad , Neoplasias Endometriales/sangre , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/mortalidad , Neoplasias de las Trompas Uterinas/tratamiento farmacológico , Neoplasias de las Trompas Uterinas/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/mortalidad , Pronóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tasa de Supervivencia , Estudios de Validación como AsuntoRESUMEN
Early identification and prognostic stratification of delayed graft function following renal transplantation has significant potential to improve outcome. Mass spectrometry analysis of serum samples, before and on day 2 post transplant from five patients with delayed graft function and five with an uncomplicated transplant, identified aminoacylase-1 (ACY-1) as a potential outcome biomarker. Following assay development, analysis of longitudinal samples from an initial validation cohort of 55 patients confirmed that the ACY-1 level on day 1 or 2 was a moderate predictor of delayed graft function, similar to serum creatinine, complementing the strongest predictor cystatin C. A further validation cohort of 194 patients confirmed this association with area under ROC curves (95% CI) for day 1 serum (138 patients) of 0.74 (0.67-0.85) for ACY-1, 0.9 (0.84-0.95) for cystatin C, and 0.93 (0.88-0.97) for both combined. Significant differences in serum ACY-1 levels were apparent between delayed, slow, and immediate graft function. Analysis of long-term follow-up for 54 patients with delayed graft function showed a highly significant association between day 1 or 3 serum ACY-1 and dialysis-free survival, mainly associated with the donor-brain-dead transplant type. Thus, proteomic analysis provides novel insights into the potential clinical utility of serum ACY-1 levels immediately post transplantation, enabling subdivision of patients with delayed graft function in terms of long-term outcome. Our study requires independent confirmation.
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Amidohidrolasas/sangre , Funcionamiento Retardado del Injerto/etiología , Trasplante de Riñón/efectos adversos , Adulto , Anciano , Área Bajo la Curva , Biomarcadores/sangre , Creatinina/sangre , Cistatina C/sangre , Funcionamiento Retardado del Injerto/sangre , Funcionamiento Retardado del Injerto/enzimología , Funcionamiento Retardado del Injerto/terapia , Supervivencia sin Enfermedad , Diagnóstico Precoz , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Estudios Longitudinales , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Proteómica/métodos , Curva ROC , Diálisis Renal , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del TratamientoRESUMEN
AIM OF THE STUDY: Validated molecular biomarkers are urgently required in colon cancer (CC) to accurately define prognosis and, ideally, to predict response to therapeutic modalities such as adjuvant chemotherapy. We aimed to identify and characterise a novel membrane-associated protein in CC tissues which may offer diagnostic and, potentially, therapeutic targeting opportunities. METHODS: Label-free mass spectrometric (MS) quantitation was employed to profile matched colon tissues for malignancy-associated proteins. The putative diagnostic utility of a chosen marker was evaluated using immunohistochemistry (IHC) on 367 CC tissue samples contained within the NCI Progression Colon Cancer tissue microarray (TMA) set. RESULTS: Retinoic acid-induced protein 3 (RAI3) was initially identified as a plasma membrane protein overexpressed in CC. Cancer-associated RAI3 over-expression was confirmed by RAI3 IHC. Although RAI3 IHC expression patterns were variable within neoplastic epithelium, 76% (n=236) of interpretable CC cases (n=312) displayed diffuse cytoplasmic expression. Of note, a sub-set of CC tissues (n=23, 7.4%) displayed very strong cytoplasmic expression, a feature significantly associated with disease recurrence in Dukes' A-C (stage I-III) patients (hazard ratio (HR)=3.076, [95%confidence interval (CI)=1.738-5.445]; p<0.001) when compared to low or negative expression of RAI3. This association retained univariate significance in Dukes' B/stage II patients only (HR=3.494, [95%CI=1.197-10.20]; p<0.022). Significantly, the prognostic capacity of RAI3 was maintained in the stage I-III cohort following multivariate modelling (HR=2.11, [95%CI 1.109-4.017], p=0.023). CONCLUSION: RAI3 is a putative prognostic marker that identifies a small subset of CC patients with high recurrence risk. This study demonstrates the potential value of modern proteomic technology in clinically relevant applications.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias del Colon/química , Receptores Acoplados a Proteínas G/análisis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/metabolismo , Quimioterapia Adyuvante , Neoplasias del Colon/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Ratones , Persona de Mediana Edad , Pronóstico , Proteómica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
PURPOSE: Protein profiling of formalin-fixed paraffin-embedded (FFPE) tissues has enormous potential for the discovery and validation of disease biomarkers. The aim of this study was to systematically characterize the effect of length of time of storage of such tissue blocks in pathology archives on the quality of data produced using label-free MS. EXPERIMENTAL DESIGN: Normal kidney and clear cell renal cell carcinoma tissues routinely collected up to 10 years prior to analysis were profiled using LC-MS/MS and the data analyzed using MaxQuant. Protein identities and quantification data were analyzed to examine differences between tissue blocks of different ages and assess the impact of technical and biological variability. RESULTS: An average of over 2000 proteins was seen in each sample with good reproducibility in terms of proteins identified and quantification for normal kidney tissue, with no significant effect of block age. Greater biological variability was apparent in the renal cell carcinoma tissue, possibly reflecting disease heterogeneity, but again there was good correlation between technical replicates and no significant effect of block age. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that archival storage time does not have a detrimental effect on protein profiling of FFPE tissues, supporting the use of such tissues in biomarker discovery studies.
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Biomarcadores/análisis , Formaldehído/química , Riñón/química , Adhesión en Parafina/métodos , Proteoma/análisis , Adulto , Anciano , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/química , Cromatografía Liquida , Femenino , Humanos , Neoplasias Renales/química , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Proteómica , Factores de TiempoRESUMEN
There are only few reports on protein products originating from overlapping mammalian genes even though computational predictions suggest that an appreciable fraction of mammalian genes could potentially overlap. Mass spectrometry-based proteomics has now acquired the tools to probe proteins in an unbiased manner, providing direct evidence of the output of the genomic and gene expression machinery. In particular, proteomics can refine gene predictions and discover novel gene-processing events and gene arrangements. Here, we report the mass spectrometric discovery and biochemical validation of the novel protein encoded by a gene overlapping rab34 oncogene. The novel protein is highly conserved in mammals. In humans, it contains 13 distinct Nine-Amino acid Residue-Repeats (NARR) with the consensus sequence PRVIV(S/T)PR in which the serine or threonine residues are phosphorylated during M-phase. NARR is ubiquitously expressed and resides in nucleoli where it colocalizes with ribosomal DNA (rDNA) gene clusters. Its distribution only partially overlaps with upstream binding factor, one of the main regulators of RNA Polymerase I activity, and is entirely uncoupled from it in mitotic cells and upon inhibition of transcription. NARR only partially colocalizes with fibrillarin, the pre-ribosomal RNA-processing protein, positioning NARR in a separate niche within the rDNA cluster.
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Nucléolo Celular/química , Proteínas Nucleares/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/análisis , Genes de ARNr , Humanos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oncogenes , Factores de Iniciación de Péptidos/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/análisis , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de Aminoácido , Proteínas de Unión al GTP rab/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2-D DIGE ('top-down') and LC-MS/MS ('bottom-up'). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high-throughput 1-D-LC-MS/MS approach, including known biomarkers and moderate-lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2-D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC-MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label-free analysis, greater run-to-run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1-D nano-LC-LTQ Orbitrap Velos analysis in a moderate through-put biomarker discovery process.
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Biomarcadores/análisis , Proteínas Sanguíneas/química , Técnicas de Inmunoadsorción , Proteómica/métodos , Biomarcadores/química , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/aislamiento & purificación , Cromatografía Liquida , Bases de Datos de Proteínas , Humanos , Isoformas de Proteínas , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO(2)-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12,035 phosphorylation sites on 4579 brain proteins of which 8446 are novel. Gene Ontology annotation reveals that 23% of identified sites are located on plasma membrane proteins, including a large number of ion channels and transporters. Together with the glycosylation sites from a recent large-scale study, they can confirm or correct predicted membrane topologies of these proteins, as we show for the examples calcium channels and glutamate receptors.
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Química Encefálica , Proteínas de la Membrana/química , Fosfoproteínas/química , Proteoma/química , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Proteínas de la Membrana/clasificación , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/clasificación , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Fosfoproteínas/clasificación , Fosfoproteínas/aislamiento & purificación , Fosfotirosina/químicaRESUMEN
Membrane proteomics is challenging because the desirable strong detergents are incompatible with downstream analysis. Recently, we demonstrated efficient removal of SDS by the filter aided sample preparation method (FASP). Here we combine FASP with our previously described small-scale membrane enrichment protocol. Analysis of a single mouse hippocampus enables identification of more than 1000 membrane proteins in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, we developed a simple anion exchange fractionation method in a StageTip format. When separating peptides into six fractions, a duplicate analysis resulted in identification of 4206 proteins of which 64% were membrane proteins. This data set covers 83% of glutamate and GABA receptor subunits identified in hippocampus in the Allen Brain Atlas and adds further isoforms. The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping. We also provide a generic protocol for combining FASP with StageTip-based ion exchange fractionation, which is generally applicable to proteome analysis.
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Hipocampo/química , Proteínas de la Membrana/análisis , Proteoma/análisis , Proteómica/métodos , Animales , Fraccionamiento Químico , Cromatografía Liquida , Intercambio Iónico , Métodos , Ratones , Espectrometría de Masas en TándemRESUMEN
Linker histones H1 are key modulators of chromatin structure. Tightness of their binding to DNA is regulated by posttranslational modifications. In this study we have analyzed posttranslational modifications of five major variants of H1 in human tissue - H1.0, H1.2, H1.3, H1.4, and H1.5. To improve sequence coverage, tryptic peptides of H1 were separated by HPLC and the individual fractions were analyzed using a peptide on-chip implementation of nanoelectrospray (TriVersa), coupled to a linear ion trap-orbitrap hybrid instrument. For quantitative analysis of lysine methylation, ionization efficiencies of methylated and nonmethylated peptides were determined using synthetic peptides. Our analysis revealed that monomethylation of lysine residues alongside with phosphorylation of serine and threonine residues is the major modification of H1 in tissue. We found that most prominent methylation sites are in the N-terminal tail and the globular domain of H1. In the C- terminal domains we identified only few and less abundant methylation sites. Quantitative analysis revealed that up to 25% of H1.4 is methylated at K-26 in human tissues. Another prominent methylation site was mapped to K-27 in H1.5, which resembles the K-26 site in H1.4. In H1.0 five less abundant (<1% of H1.0) sites were identified. Analysis of patient matched pairs of cancer and adjacent normal breast demonstrated high variation in H1 methylation between individuals.
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Neoplasias de la Mama/metabolismo , Mama/metabolismo , Histonas/química , Lisina/química , Mapeo Peptídico/métodos , Proteómica/métodos , Anciano , Mama/química , Neoplasias de la Mama/química , Femenino , Histonas/metabolismo , Humanos , Modelos Lineales , Lisina/metabolismo , Metilación , Persona de Mediana Edad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry-based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
Asunto(s)
Métodos Analíticos de la Preparación de la Muestra/métodos , Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Química Encefálica , Endopeptidasas/química , Células HeLa , Humanos , Yodoacetamida/química , Hígado/química , Ratones , Mitocondrias/química , Proteoma/química , Dodecil Sulfato de Sodio/química , Espectrometría de Masas en Tándem/métodos , Ultrafiltración/métodos , Urea/químicaRESUMEN
Periviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR(L/I)amide, SPLPR(L/I)amide, SFVR(L/I)amide, AFVR(L/I)amide, and SPAFVR(L/I)amide, were identified in the central nervous system with the common -XR(L/I)amide C-terminal sequence. The exact anatomical position of 13 labeled XR(I/L)amide expressing neuron groups and numerous peptide-containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole-mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine-fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister-groups of animals such as annelids, mollusks, and insects.