Biochemical identification of natural and synthetic bovine SAA1(46-112). (A) MCF-2 was prepurified as described in the “Materials and methods” and supplemental Methods. Finally, cation exchange chromatography fractions containing bovine MCF-2 were purified to homogeneity by RP-HPLC. Proteins were eluted in an acetonitrile gradient (0% to 80%; dashed green line). The protein concentration was reflected by the absorbance at 214 nm (A214 nm; solid blue line). In all purification steps, column fractions were screened for chemotactic activity using the semiautomated MultiScreen chemotaxis assay (dilution: 1/100), in which THP-1 cells score reproducibly in a sensitive manner (solid red line with squares). (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions of the RP-HPLC fractions 35 to 53 (20 µL/lane). Proteins were visualized by silver staining. Relative molecular mass (M_r) markers are shown in the left and right lanes. The arrowhead indicates the 7- to 8-kDa protein, corresponding to MCF-2. (C) Mass spectrometric analysis of MCF-2, identified as natural SAA1(46-112). Two percent of the RP-HPLC column flow was deviated to an electrospray ion trap mass spectrometer. (D) Mass spectrometric analysis of synthetic SAA1(46-112). SAA1(46-112) was chemically synthesized using Fmoc chemistry and purified via RP-HPLC. (C-D) The averaged mass spectra, the number of charges for the detected ions, ion intensities, and corresponding mass to charge ratios (m/z) are shown. The inserts show the deconvoluted mass spectra with the M_r of the uncharged proteins calculated using the Bruker deconvolution software.