Characterization of synthetic human proinsulin and its B8 analogs by LC-MS. The MS (ESI) taken across the entire main peak is shown in the inset. Note that distinct reverse phase packings and gradients were used, as follows: A, folded wild-type proinsulin. The chromatographic separations were performed on a C4 column using a linear gradient (5–47%) of buffer B in buffer A over 42 min. B, folded [l-SerB8] proinsulin. The chromatographic separations were performed on a C8 column using a linear gradient (5–47%) of buffer B in buffer A over 42 min. C, folded [d-AlaB8] proinsulin. The chromatographic separations were performed on a C4 column using a linear gradient (9–53%) of buffer B in buffer A over 44 min. D, folded [l-AlaB8] proinsulin. The chromatographic separations were performed on a C8 column using a linear gradient (5–47%) of buffer B in buffer A over 42 min.