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

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QC 20101213
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.

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

Dahl, KennethKTH,Analytisk kemi(Swepub:kth)u1eq8inv (författare)
Karlberg, Ann-Therese,1947Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry(Swepub:gu)xkaann (författare)
Redeby, Theres,1973Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry(Swepub:gu)xredth (författare)
Emmer, ÅsaKTH,Analytisk kemi(Swepub:kth)u1czoysx (författare)
KTHAnalytisk kemi (creator_code:org_t)

Ingår i:Journal of chromatography. B: Elsevier BV878:15-16, s. 1125-11341570-02321873-376X

Av författaren/redakt...: Jacksén, Johan; Dahl, Kenneth; Karlberg, Ann-Th ...; Redeby, Theres, ...; Emmer, Åsa

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