Aldo Profumo 1 Barbara Cardinali 1, 2 Carla Cuniberti 2 Mattia Rocco 1 1 S.C. Medicina Rigenerativa, Istituto Nazionale per la Ricerca sul Cancro 2 Dipartimento di Chimica e Chimica Industriale, Università di Genova, Genova, Italy Separation of human fibrinopeptides by capillary zone electrophoresis We describe a method for the simultaneous determination of the five fibrinopeptide forms derived from the thrombin-promoted activation of human fibrinogen by capillary zone electrophoresis (CZE). The fibrinopeptide mixture was first desalted by a solid- phase extraction (SPE) step. The analysis was performed in reversed polarity in a highly cross-linked polyethylene glycol (PEG)-coated capillary with UV-light absorption detection at 200 nm. Several parameters including buffer concentration and pH, pres- ence of an organic modifier, temperature, and applied voltage, have been tested. The best separations were obtained within 20 min, utilizing a 20 mM sodium phosphate buffer without organic modifier, in the narrow 6.1–6.2 pH range, at 257C, with an applied voltage of 20kV. Quantitative analysis is made possible by the use of sheep fibrinopeptide A as an internal standard to correct for both extraction and injection errors. Keywords: Blood coagulation / Capillary electrophoresis / Fibrinogen / Fibrinopeptides / Solid- phase extraction DOI 10.1002/elps.200406132 1 Introduction The plasma protein fibrinogen, whose blood physiological concentration ranges from 3 to 4 mg/mL (,10 25 M), is a high-molecular-weight (M r 340 000) glycoprotein com- posed of three pairs of nonidentical peptide chains (2Aa, 2Bb, and 2g) connected by a series of inter- and intrachain disulfide bonds [1–3]. During the fibrinogen-fibrin conver- sion mediated by the serine protease thrombin, four small peptides, two fibrinopeptide A (fpA) and two fibrinopep- tide B (fpB), are cleaved from the N-terminal end of the Aa and Bb chains, respectively. Removal of these peptides leads to the exposure of cryptic polymerization sites on the fibrinogen molecule resulting in the rapid formation of a fibrin clot. The absolute correlation between fibrinogen activation and fp release makes their quantification an important tool to study the early events in fibrin formation. Since the Seventies, radioimmunoassay (RIA) has been widely used for the determination of both fpA [4] and fpB [5] release. RIA is highly sensitive, but several specific antibodies would be required for the discrimination in a single assay of variant fibrinopeptides having small differ- ences such as the substitution, the post-translational modification, or the deletion of a single amino acid resi- due. This limit precludes its use in the separate evaluation of the less abundant forms fpAP, fpAY, and des Arg B [6], derived from the main fps by phosphorylation of a serine, and by deletion of an N-terminal alanine or a C-terminal arginine, respectively. Several methods have been described for the determination of fps by high-perform- ance liquid chromatography (HPLC) [6, 7]. While this technique is able to distinguish every fp form including the degradation products, it however suffers from some dis- advantages. Besides the operational costs, HPLC meth- ods are time-consuming, a satisfactory separation of fps typically requiring 50–60 min. Capillary electrophoresis (CE) can be an attractive alter- native technology to both RIA and HPLC analysis. It is a powerful analytical method because of its very high col- umn efficiency (up to millions of theoretical plates) [8] and its low running expenses. Furthermore, owing to its high mass sensitivity, CE is truly a microscale technique that can be helpful when only little amounts of sample are available. CE techniques are widely used for the analysis of small peptides [9–11]. However, to the best of our knowledge, there are no examples for the separation of fps by means of this alternative technology. Although dif- ferent modes of CE analysis could be employed for the separation of peptides, including micellar electrokinetic capillary chromatography (MEKC) [12, 13] and capillary isoelectric focusing (CIEF) [14], we have chosen capillary zone electrophoresis (CZE) because of its simplicity, high separative power, ease of quantitation, and the ability to perform rapid automated analyses. Correspondence: Dr. Aldo Profumo, S.C. Medicina Rigenera- tiva, Istituto Nazionale per la Ricerca sul Cancro c/o Centro di Biotecnologie Avanzate, Largo Rosanna Benzi, 10, I-16132 Gen- ova, Italy E-mail: aldo.profumo@istge.it Fax: 139-0105737-325 Abbreviations: fp(s), fibrinopeptide(s); HFG, human fibrinogen; SFG, sheep fibrinogen 600 Electrophoresis 2005, 26, 600–609  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim