RESUMEN
Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5×106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is generally refractory to immune checkpoint blockade, although patients with genetically unstable tumors can show modest therapeutic benefit. We previously demonstrated the presence of tumor-reactive CD8+ T cells in PDAC samples. Here, we charted the tumor-infiltrating T cell repertoire in PDAC by combining single-cell transcriptomics with functional testing of T cell receptors (TCRs) for reactivity against autologous tumor cells. On the basis of a comprehensive dataset including 93 tumor-reactive and 65 bystander TCR clonotypes, we delineated a gene signature that effectively distinguishes between these T cell subsets in PDAC, as well as in other tumor indications. This revealed a high frequency of tumor-reactive TCR clonotypes in three genetically unstable samples. In contrast, the T cell repertoire in six genetically stable PDAC tumors was largely dominated by bystander T cells. Nevertheless, multiple tumor-reactive TCRs were successfully identified in each of these samples, thereby providing a perspective for personalized immunotherapy in this treatment-resistant indication.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linfocitos T CD8-positivos , Transcriptoma/genética , Receptores de Antígenos de Linfocitos T/genética , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Neoplasias PancreáticasRESUMEN
In mass spectrometry-based proteomics, heavy internal standards are used to validate target peptide detections and to calibrate peptide quantitation. Here, we report light contamination present in heavy labelled synthetic peptides of high isotopic enrichment. Application of such peptides as assay-internal standards potentially compromises the detection and quantitation especially of low abundant cellular peptides. Therefore, it is important to adopt guidelines to prevent false-positive identifications of endogenous light peptides as well as errors in their quantitation from biological samples.
Asunto(s)
Péptidos , Proteómica , Marcaje Isotópico , Isótopos , Espectrometría de Masas , Estándares de ReferenciaRESUMEN
For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation-derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC-MS3 -based targeted detection of low-abundant human leukocyte antigen (HLA) class-I-presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen-derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA-peptide complexes, while keeping high isolation yields of low-abundant target peptides. The subsequent targeted LC-MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA-A2-restricted epitope E711-19 and ten additional E7-derived peptides on the surface of HPV16-transformed cells. T-cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low-abundant candidate epitopes to be true immunotherapy targets.
Asunto(s)
Cromatografía Liquida/métodos , Epítopos de Linfocito T/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Papillomaviridae/inmunología , Fragmentos de Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Neoplasias del Cuello Uterino/metabolismo , Epítopos de Linfocito T/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Complejo CD3/genética , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Transducción de Señal/genética , Proteína Tirosina Quinasa ZAP-70/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Complejo CD3/metabolismo , Cromatografía Liquida , Bases de Datos de Proteínas , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Humanos , Células Jurkat , Espectrometría de Masas , Proteínas de la Membrana/deficiencia , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D-interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αßTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220-TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.
Asunto(s)
Activación de Linfocitos/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/genética , Linfocitos T/metabolismo , Animales , Autoantígenos/genética , Autoantígenos/inmunología , Biomarcadores/metabolismo , Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Proteínas Activadoras de GTPasa , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Activación de Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Ratones , Proteínas del Tejido Nervioso/inmunología , Cultivo Primario de Células , Unión Proteica , Proteínas Proto-Oncogénicas B-raf/inmunología , Proteína Asociada al mTOR Insensible a la Rapamicina , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunologíaRESUMEN
Recent advances in peptide-based (bottom-up) quantitative proteomics and bioinformatics have opened unprecedented opportunities for extensive investigation of cellular proteomes and their dynamics. Here we discuss two approaches currently used to investigate the global dynamics of phosphorylation based on the isolation of phosphorylated proteins or peptides. We evaluate the accuracy of these methodologies to grasp the global dynamics of phosphorylation, and we raise awareness on ambiguities inherent to these analyses. We conclude that further development of targeted approaches should prevent inaccurate conclusions about the nature of biological regulations and in particular kinase-substrate networks.
Asunto(s)
Espectrometría de Masas/métodos , Fosfopéptidos/química , Fosfoproteínas/química , Proteoma/química , Proteómica/métodos , Animales , Humanos , Fosfopéptidos/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosforilación , Proteoma/aislamiento & purificaciónRESUMEN
The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Marcaje Isotópico , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteoma/químicaRESUMEN
Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.
Asunto(s)
Activación de Linfocitos/fisiología , Proteínas de la Membrana/metabolismo , Proteoma/metabolismo , Linfocitos T/metabolismo , Humanos , Células Jurkat , Proteínas de la Membrana/inmunología , Proteoma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiología , Linfocitos T/inmunologíaRESUMEN
Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas/metabolismo , Proteómica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Células Jurkat , Ratones , Ratones Mutantes , Factores de Transcripción NFATC/metabolismo , Fosfoproteínas/genética , Fosforilación/inmunología , Proteínas/genética , Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Factor de Transcripción AP-1/metabolismo , Tirosina/metabolismoRESUMEN
T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Activación Enzimática/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Microscopía Confocal , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.
Asunto(s)
Autoinmunidad/genética , Mutación Missense , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Tirosina/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Células Cultivadas , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Fosforilación/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T , Familia-src QuinasasRESUMEN
UNLABELLED: The central proteomics facilities pipeline (CPFP) provides identification, validation, and quantitation of peptides and proteins from LC-MS/MS datasets through an easy to use web interface. It is the first analysis pipeline targeted specifically at the needs of proteomics core facilities, reducing the data analysis load on staff, and allowing facility clients to easily access and work with their data. Identification of peptides is performed using multiple search engines, their output combined and validated using state-of-the-art techniques for improved results. Cluster execution of jobs allows analysis capacity to be increased easily as demand grows. AVAILABILITY: Released under the Common Development and Distribution License at http://cpfp.sourceforge.net/. Demonstration available at https://cpfp-master.molbiol.ox.ac.uk/cpfp_demo.
Asunto(s)
Proteómica/métodos , Programas Informáticos , Bases de Datos de Proteínas , Espectrometría de Masas/métodos , Péptidos/química , Proteínas/químicaRESUMEN
Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Supervivencia Celular/fisiología , Células Cultivadas , Clonación Molecular , Femenino , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Proteínas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Proteínas del Citoesqueleto , Humanos , Espectrometría de Masas , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Ingeniería de Proteínas , Huso Acromático/metabolismoRESUMEN
Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.
Asunto(s)
Proteínas Sanguíneas/análisis , Biblioteca de Péptidos , Proteoma/análisis , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Humanos , Ligandos , Espectrometría de Masas/métodos , Microesferas , Oligopéptidos/química , Ácidos Polimetacrílicos/química , SueroRESUMEN
The standard strategy for analysis by tandem mass spectrometry of protein phosphorylation at serine or threonine utilizes the neutral loss of H3PO4 (= 97.977/z) from proteolytic peptide molecular ions as marker fragmentation. Manual control of automatically performed neutral loss-based phosphopeptide identifications is strongly recommended, since these data may contain false-positive results. These are connected to the experimental neutral loss m/z error, to competing peptide fragmentation pathways, to limitations in data interpretation software, and to the general growth of protein sequence databases. The fragmentation-related limitations of the neutral loss approach cover (i) the occurrence of abundant 'close-to-98/z' neutral loss fragmentations, (ii) the erroneous assignment of a neutral loss other than loss of H3PO4 due to charge state mix-up, and (iii) the accidental occurrence of any fragment ion in the m/z windows of interest in combination with a charge-state mix-up. The 'close-to-98/z' losses comprise loss of proline (97.053/z), valine (99.068/z), threonine (101.048/z), or cysteine (103.009/z) preferably from peptides with N-terminal sequences PP, VP, TP, or CP, and loss of 105.025/z from alkylated methionine. Confusion with other neutral losses may occur, when their m/z window coincides with a 98/z window as result of a charge state mix-up. Neutral loss of sulfenic acid from oxidized methionine originating from a doubly charged precursor (63.998/2 = 31.999) may thus mimic the loss of phosphoric acid from a triply charged phosphopeptide (97.977/3 = 32.659). As a consequence of the large complexity of proteomes, peptide sequence ions may occur in one of the mass windows of H3PO4 loss around 97.977/z. Practical examples for false-positive annotations of phosphopeptides are given for the first two groups of error. The majority of these can be readily recognized using the guidelines presented in this study.
Asunto(s)
Fosfopéptidos/análisis , Fosfoproteínas/química , Espectrometría de Masas en Tándem/métodos , Ácidos Fosfóricos/análisis , Fosforilación , Serina/química , Treonina/químicaRESUMEN
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
Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.