RESUMO
The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the past decade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete removal of contaminants. This in turn offers the possibility for one to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in the presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our enhanced cross-linking mass spectrometry (eXL-MS) workflow is its high efficiency, allowing the analysis of a very low amount of material (15 µg). We also demonstrate that higher-energy collision dissociation (HCD) outperforms electron-transfer/higher-energy collision dissociation (EThcD) in terms of number of cross-linked peptides identified, but EThcD leads to better sequence coverage than HCD and thus easier localization of cross-linking sites.
Assuntos
Reagentes de Ligações Cruzadas/química , Complexos Multiproteicos/química , Proteínas/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Química Click , Eletroforese em Gel de Poliacrilamida , Limite de Detecção , NanotecnologiaRESUMO
OBJECTIVE: To compare the prognosis during hospitalization and maximum follow-up of 4 years in patients with myocardial infarction complicated with cardiogenic shock. METHOD: Prospective observational study practiced in a coronary Care Unit managed by cardiologists. We included patients with myocardial infarction complicated with cardiogenic shock who received early coronary revascularization. Patients were divided into two groups: older than 75 years (group A) and lower (group B), and we compared the evolution during hospitalization and maximum follow-up of 4 years. Primary end point was mortality rate in the maximum follow-up of 4years. Secondary end point was mortality rate during hospitalization. RESULTS: Ninety-seven patients were included, 45% Group A. Patients of Group B were mostly men (81% vs. 57%; P=.014), diabetics (48% vs. 21%; P=0.006), and smokers (39.6% vs. 5%). Mortality rate during hospitalization was higher in Group A (54.5%) vs. 30.2% in Group B (P=.022). Mortality rate during follow-up (primary variable) was 73% in Group A vs. 38% in Group B (P=.007). CONCLUSIONS: Myocardial infarction complicated with cardiogenic shock in elderly patients is an entity with high mortality during hospitalization and continues to worsen during long term follow-up.
Assuntos
Isquemia Miocárdica/mortalidade , Choque Cardiogênico/mortalidade , Fatores Etários , Idoso , Feminino , Humanos , Masculino , Isquemia Miocárdica/complicações , Prognóstico , Estudos Prospectivos , Choque Cardiogênico/etiologia , Taxa de Sobrevida , Fatores de TempoRESUMO
Objetivo: Comparar la evolución durante el ingreso y a largo plazo de pacientes con choque cardiogénico de origen isquémico. Método: Estudio observacional prospectivo unicéntrico llevado a cabo en la unidad coronaria de un hospital terciario manejada por cardiólogos. Se incluyen pacientes con choque cardiogénico de origen isquémico que recibieron revascularización coronaria precoz. Dividimos a los pacientes en 2 grupos: mayores de 75 anos (grupo A) y menores (grupo B), y comparamos la evolución durante el ingreso y en un seguimiento máximo de 4 años. El objetivo principal fue estimar la mortalidad a los 4 años. El objetivo secundario se definió como la mortalidad intrahospitalaria. Resultados: Incluimos a 97 pacientes, 44 del grupo A (45%). Los pacientes del grupo B eran con más frecuencia varones (81 vs. 57%, p = 0.014), diabéticos (49 vs. 21%, p: 0.006) y fumadores (39.6 vs. 4.5%, p < 0.05). La mortalidad hospitalaria fue superior en el grupo A (54.5 vs. 30.2%, p = 0.022). El objetivo principal ocurrió en 32 pacientes del grupo A frente a 20 del grupo B (73 vs. 38%, p = 0.007). Conclusión: El choque cardiogénico de origen isquémico en paciente mayores de 75 anos presenta una alta mortalidad durante la estancia hospitalaria y en el seguimiento a largo plazo.
Objective: To compare the prognosis during hospitalization and maximum follow-up of 4 years in patients with myocardial infarction complicated with cardiogenic shock. Method: Prospective observational study practiced in a coronary Care Unit managed by cardiologists. We included patients with myocardial infarction complicated with cardiogenic shock who received early coronary revascularization. Patients were divided into two groups: older than 75 years (group A) and lower (group B), and we compared the evolution during hospitalization and maximum follow-up of 4 years. Primary end point was mortality rate in the maximum follow-up of 4years. Secondary end point was mortality rate during hospitalization. Results: Ninety-seven patients were included, 45% Group A. Patients of Group B were mostly men (81% vs. 57%; P =.014), diabetics (48% vs. 21%; P = 0.006), and smokers (39.6% vs. 5%). Mortality rate during hospitalization was higher in Group A (54.5%) vs. 30.2% in Group B(P = .022). Mortality rate during follow-up (primary variable) was 73% in Group A vs. 38% in Group B (P =.007). Conclusions: Myocardial infarction complicated with cardiogenic shock in elderly patients is an entity with high mortality during hospitalization and continues to worsen during long term follow-up.
