RESUMEN
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.
Asunto(s)
Enzimas/metabolismo , Cromatografía Líquida de Alta Presión , Enzimas/química , Cinética , Mapeo Peptídico , Proteínas Tirosina Fosfatasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
MALDI mass spectrometry is explored as a method for hemoglobin characterization. To simplify and expedite the analysis, hemoglobin is obtained without purification directly from whole human blood. The use of trypsinactivated bioreactive MALDI probes is evaluated as a means to further reduce the analysis time from hours to minutes. Moreover, variations of the MALDI matrix preparation facilitate detection of the problematic tryptic peptides alpha T12, alpha T13, and beta T12. The results reveal that MALDI-based methods are easily implemented, are rapid, and allow detection of traditionally elusive tryptic peptides.
Asunto(s)
Hemoglobinas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Mapeo Peptídico/métodos , Conformación ProteicaAsunto(s)
Aldehídos/metabolismo , Proteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Bradiquinina/química , Bradiquinina/metabolismo , Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Proteínas/química , Sustancia P/química , Sustancia P/metabolismoRESUMEN
Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) is applied to the analysis of whole human blood. No separation or purification steps are required to observe individual globins and the sensitivity of the method is excellent. Mass accuracy and precision are 0.01% using a linear TOF system with pulsed ion extraction. Sickle-cell hemoglobin is easily distinguishable from normal hemoglobin. The speed and simplicity of the method make it potentially attractive for large-scale population screening.
Asunto(s)
Hemoglobinas/análisis , Hemoglobina A/análisis , Hemoglobina Falciforme/análisis , Heterocigoto , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mass spectra of single-stranded DNA oligonucleotides were acquired using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Resolution enhancement using space-velocity correlation focusing allows for facile observation of different oligomers, the direct observation of individual DNA-metal adducts, and investigation of counter ion structure.