FIG. 2.

Purification and identification of CXCL12 as chemotactic activator for HPCs in human plasma. To screen for CXCR4 stimulating activities, aliquots of fractions from different peptide libraries were screened for calcium flux-inducing activity on CXCR4-transfected HEK293T cells. The calcium flux-inducing activity was purified in four chromatographic steps. (A) Preparative RP chromatography using a Bakerbond cartridge RP-C18 column (47 mm i.d.×300 mm, 15–30 μm, 300 Å; Vydac, Hesperia, CA) with an acetonitrile gradient. (B) RP chromatography using a Bakerbond cartridge (47 mm i.d.×300 mm) with an acetonitrile gradient from 100% solvent A (10 mM HCl) to 28% solvent B (10 mM HCl in 80% acetonitrile) over 2 min, from 28% to 55% solvent B over 50 min, and from 55% to 100% solvent B over 1 min. Fractions with a volume of 50 mL were collected. (C) Analytical cation exchange chromatography using a Parcosil ProKat column (4 mm i.d.×50 mm, 7 μm, 300 Å; Biotek, Östringen, Germany) at a flow rate of 1 mL/min using a linear binary gradient of 0% solvent A (10 mM Na₂ HPO₄, pH 4.5) to 70% solvent B (10 mM Na₂ HPO₄, pH 4.5, 1 M NaCl) over 70 min, and from 70% to 100% solvent B over 5 min. Two-milliliter fractions were collected. (D) Analytical RP chromatography using a YMC RP-C18 column (4.6 mm i.d.×250 mm) at a flow rate of 0.5 mL/min using a linear binary gradient of 80% solvent A (10 mM HCl) to 60% solvent B (10 mM HCl in 80% acetonitrile) over 60 min. Fractions with a volume of 0.5 mL were collected. (E) Identification of CXCL12 in the biologically active fraction 27 of purification step D by MALDI-MS. The mass peaks marked by the arrows correspond to molecules that represent C-terminally (Lys₈₉ truncated) and N-terminally truncated CXCL12 molecules. The suggested CXCL12 molecules were confirmed by N-terminal Edman degradation. HEK, human embryonic kidney; i.d., inner diameter; MALDI-MS, matrix-assisted laser desorption/ionization–mass spectrometer.