RESUMO
OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Ensaios de Triagem em Larga Escala , Imunoensaio/métodos , Espectrometria de Massas/métodos , Doença de Alzheimer/sangue , Doenças Cardiovasculares/sangue , Transtornos do Crescimento/sangue , Humanos , Neoplasias/sangue , Insuficiência Renal/sangueRESUMO
Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.
Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Histonas/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Antibióticos Antineoplásicos , Bleomicina , Western Blotting , Senescência Celular/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosAssuntos
Algoritmos , Cromatografia Líquida/métodos , Epigênese Genética , Histonas/genética , Células-Tronco Mesenquimais/química , Processamento de Proteína Pós-Traducional/genética , Espectrometria de Massas em Tandem/métodos , Envelhecimento/fisiologia , Biologia Computacional/métodos , Metilação de DNA/genética , Enzimas/genética , HumanosRESUMO
Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.