Optimization of separation and digestion conditions in immune complexome analysis
| Autor: | Miyako Baba, Kaname Ohyama, Naoya Kishikawa, Naotaka Kuroda |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Proteomics
Gradient elution time Molecular Sequence Data Microwave-assisted digestion Biophysics Nano-liquid chromatography–tandem mass spectrometry Antigen-Antibody Complex Mass spectrometry Biochemistry Sample preparation in mass spectrometry Immune system Capillary column Antigen Tandem Mass Spectrometry Animals Humans Trypsin Amino Acid Sequence Horses Microwaves Molecular Biology Immune complexome analysis Tryptic digestion Chromatography Chemistry Model protein A protein Cell Biology Gradient elution Chromatography Liquid |
| Zdroj: | Analytical Biochemistry. 443:181-186 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2013.08.026 |
| Popis: | Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS). To improve this method, we initially investigated the effects of two factors—the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)—on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25–400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time. Analytical Biochemistry, 443(2), pp.181-186; 2013 |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect