کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1215115 1494152 2010 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates
چکیده انگلیسی

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein–hapten binding in the skin, is presented. Mass spectra of BSA–FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 878, Issues 15–16, 1 May 2010, Pages 1125–1134
نویسندگان
, , , , ,