Figure 4

Hemolymph fractionation. (a) Hemolymph from fourth- and fifth-instar larvae was fractionated by strong anion exchange using a step gradient of increasing salt. Each fraction (A-F from 0.04 M to 0.24 M salt in 0.04 M increments, plus DS = desalted hemolymph, and FT = flowthrough) was normalized by protein concentration and was subjected to a PO assay (see Methods). Activity is represented by relative reaction rates to DS (left axis, bars). Using mass spectrometry, proPO levels were measured relative to the fraction containing the highest activity (Fraction D), shown on a natural log scale (N = 3). This was accomplished by averaging the ratio of at least two peptides for proPO in a differentially label mixture of peptides from Fraction F versus the remaining fractions. Error bars = 2 standard deviations. As an example in (b), two spectra of the differentially labeled (+28Da and +32Da) peptide FSDTIVPR is shown at a 1:1 mixture of peptides from (I) Fraction D and sample DS and (II) Fraction D and E.