Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin-fluorescein isothiocyanate conjugates.
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.
Jacksén
Johan
J
Division of Analytical Chemistry, Department of Chemistry, School of Chemical Science and Engineering, Royal Institute of Technology (KTH), Teknikringen 36, SE-10044 Stockholm, Sweden.
Dahl
Kenneth
K
Karlberg
Ann-Therese
AT
Redeby
Theres
T
Emmer
Asa
A
D000485:Allergens / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000485:Allergens; D010446:Peptide Fragments; D011506:Proteins; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D002918:Chymotrypsin; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate