RESUMEN
Protein mass spectrometry (MS) is an indispensable tool to detect molecular signatures that can be associated with cellular dysregulation and disease. Despite its huge success in the life sciences, where it has led to novel insights into disease mechanisms and the identification of potential protein biomarkers, protein MS is rarely used for clinical protein assays. While conventional matrix-assisted laser desorption/ionization (MALDI) MS is not compatible with complex samples, liquid chromatography-MS (LC-MS)-based assays may be too complex and may lack the robustness and ease of automation required for routine use in the clinic. Therefore, clinical protein assays are dominated by immunohistochemistry and immunoassays which, however, often lack standardization and fully depend on antibody specificity. Immuno-MALDI (iMALDI) MS may overcome these hurdles by utilizing anti-peptide antibodies for the specific enrichment of targeted analytes and on-target detection of the captured analytes, thus combining the unique properties of MS for the unambiguous detection and quantitation of analytes with a workflow that can be fully automated. Here we discuss the requirements for clinical protein assays, the pitfalls of existing methods, how iMALDI has been successfully used to quantify endogenous peptides and proteins from clinical samples, as well as its potential as a powerful tool for companion diagnostics in the light of precision medicine.
Asunto(s)
Técnicas y Procedimientos Diagnósticos , Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Cromatografía Liquida , Humanos , Péptidos , Espectrometría de Masas en TándemRESUMEN
Motivation: In the new release of pymzML (v2.0), we have optimized the speed of this established tool for mass spectrometry data analysis to adapt to increasing amounts of data in mass spectrometry. Thus, we integrated faster libraries for numerical calculations, improved data retrieving algorithms and have optimized the source code. Importantly, to adapt to rapidly growing file sizes, we developed a generalizable compression scheme for very fast random access and applied this concept to mzML files to retrieve spectral data. Results: pymzML performs at par with established C programs when it comes to processing times. However, it offers the versatility of a scripting language, while adding unprecedented fast random access to compressed files. Additionally, we designed our compression scheme in such a general way that it can be applied to any field where fast random access to large data blocks in compressed files is desired. Availability and implementation: pymzML is freely available on https://github.com/pymzML/pymzML under GPL license. pymzML requires Python3.4+ and optionally numpy. Documentation available on http://pymzml.readthedocs.io.
Asunto(s)
Compresión de Datos/métodos , Espectrometría de Masas/métodos , Programas Informáticos , Algoritmos , Proteómica/métodosRESUMEN
Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.
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Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Regulación Leucémica de la Expresión Génica , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Clorhidrato de Bendamustina , Ligando de CD40/genética , Ligando de CD40/metabolismo , Hipoxia de la Célula , Células Cultivadas , Resistencia a Antineoplásicos , Activación Enzimática , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Compuestos de Mostaza Nitrogenada/farmacología , Oxígeno/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Vidarabina/análogos & derivados , Vidarabina/farmacología , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown. OBJECTIVE: To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation. METHODS: The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay. RESULTS: S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation. CONCLUSION: Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Plaquetas/metabolismo , Señalización del Calcio , AMP Cíclico/metabolismo , Activación Enzimática , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos/metabolismo , Agregación Plaquetaria , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
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Apoptosis/genética , Regulación de la Expresión Génica , Factor 1 de Elongación Peptídica/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Animales , Células COS , Chlorocebus aethiops , Humanos , Modelos Moleculares , Mutación , Factor 1 de Elongación Peptídica/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Platelets are anucleated cells and therefore ideal research objects for modern proteome analyses. Despite their importance in thrombosis and hemostasis the protein content of platelets is still poorly characterized in major parts. In preparation for bioinformatic and functional studies a series of proteomic analyses was conducted for platelet subproteomes as well as for posttranslational modifications. Thereby, the identification of 489 proteins, over 550 phosphorylations and 326 N-glycosylation sites was possible, which were not identified in previous proteome studies of platelets. Those results represent new research possibilities for functional characterization of platelet proteins as well as their modifications.
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Plaquetas/fisiología , Proteínas Sanguíneas/genética , Proteoma , Proteómica/métodos , Proteínas Sanguíneas/metabolismo , Hemostasis , Humanos , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Procesamiento Proteico-Postraduccional , Trombosis/sangreRESUMEN
BACKGROUND: We tested the hypothesis that serum apolipoprotein H (apo H) concentration increases after an oral fat load. Such a study would give valuable insight into whether apo H was influenced by the postprandial state. METHODS: Ten male subjects aged 24-48 years were fed 62.5 g of total fat (saturates 12 g, monounsaturates 35.3 g, polyunsaturates 12.5 g). Venous blood was sampled hourly for 5 h post-oral fat load. RESULTS: No significant change in serum apo H concentration occurred following the oral fat load. However, serum apo H in the baseline samples correlated significantly with subject body mass index (r = 0.683, P < 0.05), body fat mass (r = 0.778, P < 0.01), lean body mass (r = 0.693, P < 0.05), serum triglyceride (r = 0.732, P < 0.02), serum insulin (r = 0.808, P < 0.01) and insulin resistance index (r = 0.794, P < 0.01). In stepwise multiple linear regression model, with serum apo A1, apo B, lipoprotein(a), total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, plasma glucose and insulin and apo H as the dependent variable, insulin remained in the model (r = 0.81, P < 0.01). Conversely, with body mass index, body fat mass, lean body mass and waist/hip ratio in the model and apo H as dependent variable, only body fat mass remained in the model (r = 0.78, P < 0.01). CONCLUSIONS: Serum apo H may be involved in insulin resistance and relates to various indices of adipose tissue, including body fat mass. However, serum apo H concentrations do not significantly change postprandially.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Glicoproteínas/sangre , Adulto , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Grasas de la Dieta/efectos adversos , Humanos , Insulina/sangre , Resistencia a la Insulina , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Modelos Biológicos , beta 2 Glicoproteína IRESUMEN
BACKGROUND: Elevated serum total sialic acid (TSA) has been shown to be associated with increased cardiovascular mortality. It has been postulated that atherogenesis is a postprandial phenomenon. We tested the hypothesis that serum TSA and other acute phase proteins, namely C-reactive protein (CRP) and fibrinogen, may be related to the postprandial state. METHODS: Ten healthy male subjects, aged 24-48 years, were fed 62.5 g of total fat (saturates 12 g, monounsaturates 35.3 g and polyunsaturates 12.5 g) in the form of strawberry flavoured Calogen. Venous blood was sampled hourly for 5 h. Concentrations of serum triglyceride, TSA and acute phase proteins were measured. RESULTS: Serum triglyceride concentration increased postprandially, peaking at 240 min. Serum CRP and plasma fibrinogen did not significantly increase after the oral fat load. However, serum TSA did increase from baseline (0.599+/-0.051 g/l) in response to the oral fat load, peaking at 120 min post-oral fat load (0.633+/-0.066 g/l, P<0.02). There was a significant correlation between serum TSA and plasma fibrinogen at baseline (rho=0.62, P=0.05) but not for serum CRP (rho=-0.22) or triglyceride (rho=0.21). CONCLUSIONS: We conclude that serum TSA increases postprandially and this finding gives further insight as to why the former is considered to be a cardiovascular risk factor.
RESUMEN
Previous analysis of a naturally occurring C1 inhibitor P2 mutant (Ala(443)-->Val) indicated a role for P2 in specificity determination. To define this role and that of other reactive center loop residues, a number of different amino acids were introduced at P2, as well as at P6 (Ala(439)) and P8'/9' (Gln(452)Gln(453)). Ala(439)-->Val is a naturally occurring mutant observed in a patient with hereditary angioedema. Previous data suggested that Gln(452)Gln(453) might be a contact site for C1s. Reactivity of the inhibitors toward target (C1s, C1r, kallikrein, beta factor XIIa, and plasmin) and nontarget proteases (alpha-thrombin and trypsin) were studied. Substitution of P2 with bulky or charged residues resulted in decreased reactivity with all target proteases. Substitution with residues with hydrophobic or polar side chains resulted in decreased reactivity with some proteases, but in unaltered or increased reactivity with others. Second order rate constants for the reaction with C1s were determined for the mutants with activities most similar to the wild-type protein. The three P2 mutants showed reductions in rate from 3.35 x 10(5) M(-1)s(-1) for the wild type to 1.61, 1.29, and 0.63 x 10(5) for the Ser, Thr, and Val mutants, respectively. In contrast, the Ala(439)-->Val and the Gln(452)Gln(453)-->Ala mutants showed little difference in association rates with C1s, in comparison with the wild-type inhibitor. The data confirm the importance of P2 in specificity determination. However, the P6 position appears to be of little, if any, importance. Furthermore, it appears unlikely that Gln(452)Gln(453) comprise a portion of a protease contact site within the inhibitor.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Inactivadoras del Complemento 1/metabolismo , Fragmentos de Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Sustitución de Aminoácidos/genética , Aminoácidos/genética , Animales , Células COS , Proteínas Inactivadoras del Complemento 1/genética , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Factor XIIa/metabolismo , Fibrinolisina/metabolismo , Calor , Humanos , Calicreínas/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Desnaturalización Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Trombina/metabolismo , Tripsina/metabolismoAsunto(s)
Isquemia/etiología , Pancreatitis Alcohólica/complicaciones , Enfermedades de la Retina/etiología , Enfermedad Aguda , Adulto , Angiografía con Fluoresceína , Humanos , Mácula Lútea/irrigación sanguínea , Masculino , Pancreatitis Alcohólica/diagnóstico , Enfermedades de la Retina/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
The C5a receptor is expressed by a variety of cell types. These studies demonstrate by immunohistochemistry that the receptor is present on the surface of proximal and distal tubular epithelial cells from normal kidney. In addition, the receptor was detected on transitional epithelial cells of the ureter and bladder. Primary proximal tubular cultures and a proximal tubular cell line both also expressed the C5a receptor, as demonstrated by immunofluorescence and by FACS analysis. The presence of mRNA encoding the receptor was confirmed by reverse transcriptase-polymerase chain reaction analysis. As opposed to its effect on glomerular mesangial cells, the receptor did not mediate a proliferative response by the proximal tubular cells. C5a also did not enhance the synthesis/secretion of transforming growth factor-beta 1, monocyte chemoattractant protein-1, platelet-derived growth factor-AB or tumour necrosis factor-alpha by cultured proximal tubular cells. Therefore, although the C5a receptor clearly is expressed by proximal tubular cells, clarification of its functional relevance on this cell type awaits further studies.
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Antígenos CD/biosíntesis , Túbulos Renales Proximales/metabolismo , Receptores de Complemento/biosíntesis , Antígenos CD/análisis , Antígenos CD/genética , División Celular , Células Cultivadas , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Mesangio Glomerular/citología , Sustancias de Crecimiento/análisis , Humanos , Túbulos Renales Distales/química , Túbulos Renales Proximales/citología , Músculo Liso/química , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Receptor de Anafilatoxina C5a , Receptores de Complemento/análisis , Receptores de Complemento/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937/metabolismo , Uréter/química , Vejiga Urinaria/químicaRESUMEN
A dysfunctional C1 inhibitor (C1 INH) from a family in whom the propositus presented with systemic lupus erythematosus but without angioedema previously was shown to have diminished inhibitory activity toward isolated C1r and C1s, and intact C1. The mutation was identified as replacement of Ala443 (P2) with Val. This study further analyzed the reactivity of this mutant and characterized two mutants with Ser or Asp at this position. Ser at P2 does not interfere with binding of target proteases. However, the mutant with Asp at this position is unable to bind C1r and beta factor XIIa, and also has a decreased rate of reaction with C1s and kallikrein. Therefore, alteration of polarity alone had no effect on binding, while a bulky and/or charged side chain was not tolerated. Although defective in inhibition of C1r and C1s, the P2 A-->V mutant had acquired the ability to complex with trypsin. It also completely retained the ability to complex with kallikrein and factor XIIa. None of the 10 individuals expressing this mutant protein has ever had angioedema. This observation, combined with normal inhibition of contact system proteases and defective inhibition of complement proteases, suggests that angioedema is caused by bradykinin generated from contact system activation.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteínas Inactivadoras del Complemento 1/genética , Alanina/genética , Animales , Sitios de Unión , Células COS , Proteínas Inactivadoras del Complemento 1/metabolismo , Proteínas Inactivadoras del Complemento 1/farmacología , Proteína Inhibidora del Complemento C1 , Humanos , Mutagénesis Sitio-Dirigida , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacologíaRESUMEN
Dysfunctional C1 inhibitor (C1INH)-Ta is a naturally occurring mutant from a patient with type II hereditary angioedema. This mutant has a deletion of the codon for Lys-251, which is located in the connecting strand between helix F and strand 3A, overlying beta sheet A. Deletion of this Lys modifies the amino acid sequence at this position from Asn-Lys-Ile-Ser to Asn-Ile-Ser and creates a new glycosylation site. To further characterize the mechanism of dysfunction, we have analyzed the recombinant normal and Ta proteins expressed by COS cells in addition to the proteins in serum and isolated from serum. Recombinant C1INH-Ta revealed an intermediate thermal stability in comparison with the intact and reactive center cleaved normal proteins. Analysis of the reactivity of this recombinant protein with target proteases demonstrated no complex formation with C1s, C1r, or kallikrein. Inefficient complex formation was, however, clearly detectable with beta-factor XIIa. Each protease produced partial cleavage of the recombinant mutant inhibitor. Recombinant C1INH-Ta, on 7.5% SDS-polyacrylamide gel electrophoresis and by size fractionation on Superose 12, showed a higher molecular weight fraction that was compatible in size with dimer formation. However, no multimerization of C1INH-Ta isolated from serum or of C1INH-Ta in serum, was observed. The C1INH-Ta dimer expressed the epitopes that normally are expressed only on the protease complexed or the cleaved inhibitor. These epitopes were not expressed on the monomeric inhibitor. The data suggest that the mutation in C1INH-Ta results in a folding abnormality that behaves as if it consists of two populations of molecules, one of which is susceptible to multimerization and one of which is converted to a substrate, but which retains residual inhibitory activity.
Asunto(s)
Proteínas Inactivadoras del Complemento 1/química , Animales , Células COS , Cromatografía en Gel , Factor XIIa/metabolismo , Glicoproteínas/química , Glicosilación , Calor , Humanos , Lisina/química , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Conformación Proteica , Desnaturalización Proteica , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
We have determined the cause of an unusual C1 inhibitor abnormality in a large kindred. We previously found that half of serum C1 inhibitor molecules in affected kindred members are normal. The other half complexed with C1s but showed little complex formation with C1r. These molecules also appeared to be relatively resistant to digestion by trypsin. Taken together, the findings suggested that members of this kindred are heterozygous for an unusual C1 inhibitor mutation. Sequencing of genomic DNA from the kindred revealed that thymine has replaced cytosine in the codon for Ala443 (P2 residue) in one C1 inhibitor allele, resulting in substitution with a Val residue. To test the effect of this substitution, a mutant C1 inhibitor containing Ala443-->Val was constructed by site-directed mutagenesis and expressed in COS-1 cells. Both the Ala443-->Val mutant and the wild-type C1 inhibitor complexed completely with C1s, kallikrein, and coagulation Factor XIIa after incubation at 37 degrees C for 60 min. In contrast, the mutant inhibitor failed to complex completely with C1r under the same conditions. Time course analysis showed that the ability of the mutant to complex with C1s is also impaired: although it complexed completely in 60 min, the rate of complex formation during a 0-60-min incubation was decreased compared with wild-type C1 inhibitor. The mutant inhibitor also formed a complex with trypsin, a serine protease that cleaves, and is not inhibited by, wild-type C1 inhibitor. The Ala443-->Val mutation therefore converts C1 inhibitor from a substrate to an inhibitor of trypsin. These studies emphasize the role of the P2 residue in the determination of target protease specificity.