Proteinpilot software package

PDE3A and PKG-Iα constructs were transiently transfected in HEK293 cells for 48 h. PDE3A was immunoprecipitated from the cell lysates as described previously. The protein was resolved using 4 to 20% Tris–glycine SDS-PAGE and visualized by colloidal Coomassie stain (Bio-Rad Laboratories). The band corresponding to PDE3A (approximately 130 kDa) was excised and destained with 100 mM ammonium bicarbonate/50% (v/v) acetonitrile. Protein in gel was subjected to reduction and alkylation of cysteine residues before overnight in-gel digestion with chymotrypsin in 100 mM Tris–HCl containing 10 mM CaCl₂. The peptides were extracted with 0.1% TFA, 60% acetonitrile, and evaporated to near dryness. MS analysis of PDE3A was then performed as described (59 (link)). All spectra were taken on an ABSciex 5800 MALDI-TOF mass spectrometer in positive reflector mode (10 kV) with a matrix of cyano-4-hydroxycinnamic acid. At least 1000 laser shots were averaged to get each spectrum. The masses were calibrated to known peptide standards. The MS spectra were analyzed in the ProteinPilot software package (AB Sciex).