RESUMEN
The effect of vortex-induced vibration during tryptic digestion was investigated by applying different vibrational speeds (0, 600, 1200, or 2500 rpm) to digestion solutions for varying durations (10, 20, 30, 40, or 60 min) at two different incubation temperatures (25°C or 37°C). The most rapid digestion was observed with the highest vibrational speed and temperature. With the application of 2500 rpm at 37°C, the tryptic digestion of each of three standard proteins (cytochrome c, myoglobin, or bovine serum albumin) provided complete disappearance of the protein within 60 min, as determined by matrix-assisted laser desorption/ionization mass spectrometry. Compared to conventional overnight digestion, 60-min vortex-assisted tryptic digestion generated longer peptides, due primarily to the limited digestion time and provided better sequence coverages (89% vs. 78% for cytochrome c, 100% vs. 87% for myoglobin, and 38% vs. 26% for BSA). The longer peptides should be advantageous to analytical methods such as the middle-down approach that benefit from increased sequence coverage of proteins. Vortex-assisted tryptic digestion is expected to be a useful method for rapid tryptic digestion of proteins.
Asunto(s)
Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Tripsina/química , Tripsina/metabolismo , Animales , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , VibraciónRESUMEN
The effectiveness of microwave-assisted extraction of proteins from human hair samples was evaluated. Extractions were performed from 2-mg hair samples in an extraction solution consisting of 25 mM Tris-HCl (pH 8.5), 2.6 M thiourea, 5 M urea, and 5% mercaptoethanol. During extraction, samples were exposed to microwave radiation (600 W) for a specified incubation period (5-120 min). The extraction efficiency of samples that had been incubated for 60 min was similar to that of samples that had been heated at 50°C for 24 h using the conventional Shindai method.
Asunto(s)
Cabello/metabolismo , Microondas , Proteínas/química , Humanos , Queratinas/química , Queratinas/aislamiento & purificación , Proteínas/aislamiento & purificaciónRESUMEN
A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).
Asunto(s)
Fragmentos de Péptidos/química , Mapeo Peptídico/instrumentación , Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Tripsina/química , Acetonitrilos/química , Secuencia de Aminoácidos , Animales , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Caballos , Datos de Secuencia Molecular , Mioglobina/química , Mioglobina/metabolismo , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico/métodos , Presión , Proteínas/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Jeringas , Tripsina/metabolismoRESUMEN
Time-dependent reduction of the disulfide bonds of immunoglobulin G (IgG) using dithiothreitol (DTT) was used to characterize the structure of IgG. After treatment with DTT, gel electrophoresis was used to separate the resulting IgG fragments, which were subsequently quantified. Using this approach, the disulfide bond between a light chain and a heavy chain was determined to be cleaved faster than the disulfide bonds between two heavy chains.
Asunto(s)
Disulfuros/química , Inmunoglobulina G/química , Ditiotreitol/química , Oxidación-Reducción , Conformación Proteica , TemperaturaRESUMEN
Chitooligosaccharides with degrees of polymerization (DPs) 2-12 were successfully separated and characterized using LC-MS/MS analysis with an amine column and a quadrupole ion trap analyzer. Major MS peaks for each chromatographic peak originated from deacetylated chitooligosaccharides while N-acetylated chitooligosaccharides were observed as minor peaks. The ratio of acetylation per glucosamine unit for each chitooligomer was higher for oligomers with higher DPs. The MS/MS spectra of chitooligosaccharides were used to characterize the chitooligosaccharide fragmentation patterns. To the best of our knowledge, this is the first report of on-line LC-MS/MS analysis of chitooligosaccharides with DP higher than 6.