Separation of PBP truncation products generated on coincubation with PMN.
Neutrophils (1 × 107/mL) were coincubated with 2 μmol/L PBP for 10 minutes at 37° C in D-PBS and after acidification with TFA 500 μL of the cell-free supernatant (SN) was applied to an analytical C2/C18 column equilibrated in 0.1% TFA. The column was developed with a linear gradient of 17.5% to 37.5% acetonitrile in 0.1% TFA (dashed line). The profile of eluting proteins as detected at λ = 214 nm (solid line) was compared to that of SN from PMN not having received PBP, and only those peaks (1-4, shaded) that newly emerged in the SN of PBP-supplemented cells, compared with cells not having received PBP, were collected and further analyzed by mass spectroscopy. The molecular masses given above peaks 1 to 3 are representative for the respective major component detected, whereas for peak 4 the masses of the major (bold) and 2 minor components are given.
