RESUMEN
Diacyl glycerophospholipids (GPs) belong to the most abundant lipid species in living organisms and consist of a glycerol backbone with fatty acyl groups in sn-1 and sn-2 and a polar head group in the sn-3 position. Regioisomeric mixed diacyl GPs have the same fatty acyl composition but differ in their allocation to sn-1 or sn-2 of the glycerol unit. In-depth analysis of regioisomeric mixed diacyl GP species composed of fatty acyl moieties that are similar in length and degree of saturation typically requires either chemical derivatization or sophisticated analytical instrumentation, since these types of regioisomers are not well resolved under standard ultra-performance liquid chromatography (UPLC) conditions. Here, we introduce a simple and fast method for diacyl GP regioisomer analysis employing UPLC tandem mass spectrometry (MS/MS). This GP regioisomer analysis is based both on minor chromatographic retention time shifts and on major differences in relative abundances of the two fatty acyl anion fragments observed in MS/MS. To monitor these differences with optimal precision, MS/MS spectra are recorded continuously over the UPLC elution profile of the lipid species of interest. Quantification of relative abundances of the regioisomers was performed by algorithms that we have developed for this purpose. The method was applied to commercially available mixed diacyl GP standards and to total lipid extracts of Escherichia coli (E. coli) and bovine liver. To validate our results, we determined regioisomeric ratios of phosphatidylcholine (PC) standards using phospholipase A2-specific release of fatty acids from the sn-2 position of the glycerol backbone. Our results show that most analyzed mixed diacyl GPs of biological origin exhibit significantly higher regioisomeric purity than synthetic lipid standards. In summary, this method can be implemented in routine LC-MS/MS-based lipidomics workflows without the necessity for additional chemical additives, derivatizations, or instrumentation.
Asunto(s)
Cromatografía Liquida/métodos , Glicerofosfolípidos/análisis , Glicerofosfolípidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Escherichia coli/química , Glicerofosfolípidos/normas , Hígado/química , Estándares de Referencia , EstereoisomerismoRESUMEN
Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.
Asunto(s)
Citocinesis/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinesis/genética , Inmunoprecipitación , Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Mitosis/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
This review retraces the role of stable isotopes and mass spectrometry in the life sciences. The timeline is divided into four segments covering the years 1920-1950, 1950-1980, 1980-2000, and 2000 until today. For each period methodic progress and typical applications are discussed. Application of stable isotopes is driven by improvements of mass spectrometry, chromatography, and related fields in sensitivity, mass accuracy, structural specificity, complex sample handling ability, data output, and data evaluation. We currently experience the vision of omics-type analyses, that is, the comprehensive identification and quantification of a complete compound class within one or a few analytical runs. This development is driven by stable isotopes without competition by radioisotopes. In metabolic studies as classic field of isotopic tracer experiments, stable isotopes and radioisotopes were competing solutions, with stable isotopes as the long-term junior partner. Since the 1990s the number of metabolic studies with radioisotopes decreases, whereas stable isotope studies retain their slow but stable upward tendency. Unique fields of stable isotopes are metabolic tests in newborns, metabolic experiments in healthy controls, newborn screening for inborn errors, quantification of drugs and drug metabolites in doping control, natural isotope fractionation in geology, ecology, food authentication, or doping control, and more recently the field of quantitative omics-type analyses. There, cells or whole organisms are systematically labeled with stable isotopes to study proteomic differences or specific responses to stimuli or genetic manipulation. The duo of stable isotopes and mass spectrometry will probably continue to grow in the life sciences, since it delivers reference-quality quantitative data with molecular specificity, often combined with informative isotope effects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:58-85, 2017.
Asunto(s)
Isótopos , Espectrometría de Masas/métodos , Proteómica/métodos , Aminoácidos/metabolismo , Cromatografía Liquida/métodos , Óxido de Deuterio/toxicidad , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Infecciones por Helicobacter/diagnóstico , Humanos , Recién Nacido , Marcaje Isotópico/métodos , Tamizaje Neonatal , Técnica de Dilución de Radioisótopos , Radiometría , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células Eritroides/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Eritropoyetina , Transducción de Señal/fisiología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Biología Computacional , Células Eritroides/citología , Humanos , Neoplasias Pulmonares/genética , Receptores de Eritropoyetina/análisis , Receptores de Eritropoyetina/clasificación , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismoRESUMEN
N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage-specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.
Asunto(s)
Aciltransferasas/fisiología , Tolerancia Inmunológica , Ácido Mirístico/metabolismo , Proteínas/metabolismo , Linfocitos T/inmunología , Aciltransferasas/deficiencia , Animales , Antígenos CD4/análisis , Células Cultivadas , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/análisis , Proteínas de la Membrana/fisiología , Ratones , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Receptores de Antígenos de Linfocitos T/fisiologíaRESUMEN
A new method for microquantification of phospholipid classes by nanoelectrospray mass spectrometry and stable isotope dilution is presented. The method covers the sum of phosphatidylcholine and sphingomyelin and in addition selectively quantifies phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. A phospholipid class is quantified together with its corresponding lyso-species due to the presence of a common head group. Phospholipids are extracted from tissue lysates, hydrolysed by hydrofluoric acid, and the liberated polar head groups choline, ethanolamine, serine, and inositol are quantified by nanoelectrospray mass spectrometry using deuterium-labeled analogs of the head groups as internal standards. The method is applied to tissue samples of a gastrointestinal tumor and of corresponding non-affected control tissue. In the tumor sample, the abovementioned phospholipids were found at roughly threefold elevated concentrations with a virtually unaltered relative abundance profile.
RESUMEN
A new method for inorganic phosphate microquantification is introduced based on negative ion electrospray tandem mass spectrometry and stable isotope dilution by (18)O4-labeled phosphate. Quantification is performed using the non-labeled and (18)O3-labeled [P(18)O3](-) fragment ions at m/z 79 and 85, respectively, formed by dissociation of the [H2PO4](-) ion at m/z 97 and 105, respectively, visible in negative ion electrospray ionization mass spectrometry (ESI-MS) spectra. Tandem mass spectrometry was selected to remove an overlap with the isobaric [HSO4](-) ion at m/z 97 of sulfate and to establish an optimal sensitivity of the quantification assay. It is demonstrated that the assay can also measure the sum of inorganic and phosphoryl phosphate by prior enzymatic hydrolysis of phosphoryl phosphate. The assay works with phosphate concentrations in the micromolar range and, in combination with nano-ESI, is capable to quantitate absolute amounts of phosphate in the low nanogram range from complex samples.
Asunto(s)
Fosfatos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Organofosfatos/análisis , Organofosfatos/química , Isótopos de Oxígeno , Fosfatos/química , Fosfopéptidos/análisis , Sulfatos/análisis , Sulfatos/químicaRESUMEN
STAT5A and STAT5B are important transcription factors that dimerize and transduce activation signals of cytokine receptors directly to the nucleus. A typical cytokine that mediates STAT5 activation is erythropoietin (Epo). Differential functions of STAT5A and STAT5B have been reported. However, the extent to which phosphorylated STAT5A and STAT5B (pSTAT5A, pSTAT5B) form homo- or heterodimers is not understood, nor is how this might influence the signal transmission to the nucleus. To study this, we designed a concept to investigate the isoform-specific dimerization behavior of pSTAT5A and pSTAT5B that comprises isoform-specific immunoprecipitation (IP), measurement of the degree of phosphorylation, and isoform ratio determination between STAT5A and STAT5B. For the main analytical method, we employed quantitative label-free and -based mass spectrometry. For the cellular model system, we used Epo receptor (EpoR)-expressing BaF3 cells (BaF3-EpoR) stimulated with Epo. Three hypotheses of dimer formation between pSTAT5A and pSTAT5B were used to explain the analytical results by a static mathematical model: formation of (i) homodimers only, (ii) heterodimers only, and (iii) random formation of homo- and heterodimers. The best agreement between experimental data and model simulations was found for the last case. Dynamics of cytoplasmic STAT5 dimerization could be explained by distinct nuclear import rates and individual nuclear retention for homo- and heterodimers of phosphorylated STAT5.
Asunto(s)
Espectrometría de Masas/métodos , Modelos Teóricos , Multimerización de Proteína , Factor de Transcripción STAT5/química , Algoritmos , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Cromatografía Liquida , Citoplasma/metabolismo , Eritropoyetina/farmacología , Immunoblotting , Cinética , Ratones , Datos de Secuencia Molecular , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 1881-1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca(2+)-dependent exocytosis and Ca(2+)-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5'-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na(+), K(+) and Ca(2+) homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca(2+)-independent efflux of NTs increases while the Ca(2+)-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.
Asunto(s)
Aciltransferasas/deficiencia , Terminales Presinápticos/metabolismo , Transmisión Sináptica , Sinaptosomas/metabolismo , Acetilcolina/metabolismo , Aciltransferasas/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Cerebelo/metabolismo , Condrodisplasia Punctata Rizomélica/genética , Condrodisplasia Punctata Rizomélica/metabolismo , Exocitosis , Perfilación de la Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Peroxidación de Lípido , Malondialdehído/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Fosfatidiletanolaminas/metabolismo , Plasmalógenos/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
In Saccharomyces cerevisiae the Cdc14 phosphatase plays a well-established role in reverting phosphorylation events on substrates of the mitotic cyclin-dependent kinase (M-Cdk1), thereby promoting mitotic exit and downregulation of M-Cdk1 activity. Cdc14 localizes at the site of cell cleavage after M-Cdk1 inactivation, suggesting that Cdc14 may perform a crucial, yet ill-defined, role during cytokinesis. Here, we identified Inn1, as a novel direct substrate of both M-Cdk1 and Cdc14. Cdc14 colocalizes with Inn1 at the cell division site and interacts with the C-terminal proline-rich domain of Inn1 that mediates its binding to the SH3-domain-containing proteins Hof1 and Cyk3. We show that phosphorylation of Inn1 by Cdk1 partially perturbs the interaction of Inn1 with Cyk3 thereby reducing the levels of Cyk3 at the cell division site. We propose that Cdc14 counteracts Cdk1 phosphorylation of Inn1 to facilitate Inn1-Cyk3 complex formation and so promote cytokinesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Citocinesis , Activación Enzimática , Microscopía Fluorescente , Mitosis , Fosforilación , Dominios Proteicos Ricos en Prolina , Unión Proteica , Transporte de Proteínas , Imagen de Lapso de Tiempo/métodos , Técnicas del Sistema de Dos Híbridos , Dominios Homologos srcRESUMEN
Centriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the γ-tubulin ring complex (γ-TuRC) that nucleates microtubules. GCP6 is a member of the γ-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the γ-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal γ-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.
Asunto(s)
Centriolos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Centriolos/metabolismo , Centriolos/ultraestructura , Humanos , Proteínas Asociadas a Microtúbulos/fisiología , Fosforilación , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation, and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY, and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC-MS. This method enabled us to determine the abundances of phospho-forms with a relative variability of ≤5% (SD). We observed a switch-like preference of ERK phospho-form abundances toward the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multisite phosphorylation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/aislamiento & purificación , Hepatocitos/metabolismo , Quinasas Quinasa Quinasa PAM/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Secuencias de Aminoácidos , Animales , Isótopos de Carbono , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Cultivo Primario de Células , Espectrometría de Masas en TándemRESUMEN
The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación hacia Abajo/inmunología , Activación de Linfocitos/inmunología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Células COS , Chlorocebus aethiops , Humanos , Células Jurkat , Fosforilación , Unión Proteica/inmunología , Serina/metabolismo , Linfocitos T/metabolismoRESUMEN
This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site-specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry-based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site-specific degrees of phosphorylation including the label-free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.
Asunto(s)
Fosfopéptidos/análisis , Proteínas/química , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida/métodos , Humanos , Marcaje Isotópico/métodos , Datos de Secuencia Molecular , FosforilaciónRESUMEN
A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one-to-one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI- and MALDI-MS.
Asunto(s)
Péptidos/química , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Tripsina/metabolismoRESUMEN
Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.
Asunto(s)
Janus Quinasa 2/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Animales , Apoptosis , Clonación Molecular , Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Femenino , Etiquetado Corte-Fin in Situ , Janus Quinasa 2/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Análisis por Micromatrices , Modelos Biológicos , Fosforilación , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance (p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio.
Asunto(s)
Calmodulina/química , Marcaje Isotópico/métodos , Proteómica/métodos , Secuencia de Aminoácidos , Calmodulina/genética , Calmodulina/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Activation of the NF-kappaB pathway in T cells is required for induction of an adaptive immune response. Hematopoietic progenitor kinase (HPK1) is an important proximal mediator of T-cell receptor (TCR)-induced NF-kappaB activation. Knock-down of HPK1 abrogates TCR-induced IKKbeta and NF-kappaB activation, whereas active HPK1 leads to increased IKKbeta activity in T cells. Yet, the precise molecular mechanism of this process remains elusive. Here, we show that HPK1-mediated NF-kappaB activation is dependent on the adaptor protein CARMA1. HPK1 interacts with CARMA1 in a TCR stimulation-dependent manner and phosphorylates the linker region of CARMA1. Interestingly, the putative HPK1 phosphorylation sites in CARMA1 are different from known PKC consensus sites. Mutations of residues S549, S551, and S552 in CARMA1 abrogated phosphorylation of a CARMA1-linker construct by HPK1 in vitro. In addition, CARMA1 S551A or S5549A/S551A point mutants failed to restore HPK1-mediated and TCR-mediated NF-kappaB activation and IL-2 expression in CARMA1-deficient T cells. Thus, we identify HPK1 as a kinase specific for CARMA1 and suggest HPK1-mediated phosphorylation of CARMA1 as an additional regulatory mechanism tuning the NF-kappaB response upon TCR stimulation.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Guanilato Ciclasa/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/metabolismo , Animales , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD/genética , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Guanilato Ciclasa/genética , Humanos , Inmunoprecipitación , Células Jurkat , Mutación , FN-kappa B/genética , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , TransfecciónRESUMEN
Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.
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Eritropoyetina , Sobrecarga de Hierro , Eritropoyesis/fisiología , Eritropoyetina/farmacología , Femenino , Hemo , Hemoglobinas , Humanos , Hierro/metabolismo , Embarazo , Proteoma , AzufreRESUMEN
It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase µLC-ICP-MS or nanoLC-ESI-MS. Using µLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.