Detection of transferrin isoforms in human serum: comparison of UV and ICP-MS detection after CZE and HPLC separations.
Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L(-1) borate buffer (pH 8.4) containing 3 mmol L(-1) diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm x 50 microm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L(-1) in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP-MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP-MS instrument was used to minimize polyatomic interferences on the (56)Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02-0.04 micromol L(-1) Tf for HPLC-ICP (ORS)-MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S(2), S(3), S(4), S(5), and S(6)) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.
Arizaga Rodríguez
Sandra
S
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain.
Blanco González
Elisa
E
Alvarez Llamas
Gloria
G
Montes-Bayón
Maria
M
Sanz-Medel
Alfredo
A
D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D011247:Pregnancy; D020033:Protein Isoforms; D014168:Transferrin / Q000032:analysis*
D020033:Protein Isoforms; D014168:Transferrin