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Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin fluorescein isothiocyanate conjugates

Jacksén, Johan, (författare)
KTH, Analytisk kemi
Dahl, Kenneth, (författare)
KTH, Analytisk kemi
Karlberg, Ann-Therese (författare)
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Redeby, Theres (författare)
Emmer, Åsa, (författare)
KTH, Analytisk kemi
Emmer, Asa (författare)
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KTH Skolan för kemivetenskap (CHE). Kemi. Analytisk kemi. 
Göteborgs universitet Naturvetenskapliga fakulteten. Institutionen för kemi. 
2010
Engelska.
Ingår i: Journal of chromatography. B. - 1570-0232. ; 878:15-16, s. 1125-1134
  • Tidskriftsartikel (refereegranskat)
Abstract Ämnesord
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  • 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 m M. 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. (C) 2010 Elsevier B.V. All rights reserved.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)
NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Nyckelord

NATURAL SCIENCES Chemistry Analytical chemistry
NATURVETENSKAP Kemi Analytisk kemi
Capillary electrophoresis
MALDI-TOF-MS
Contact allergy
Peptide-fluorescein isothiocyanate adducts
Bovine serum albumin
Kemi
Chemistry
Capillary electrophoresis; MALDI-TOF-MS; Contact allergy; Peptide-fluorescein isothiocyanate adducts; Bovine serum albumin

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