Peptide mapping and disulfide bond analysis of myeloid progenitor inhibitory chemokine and keratinocyte growth factor by matrix-assisted laser desorption ionization mass spectrometry

Peptide mapping and matrix-assisted laser desorption ionization mass spectr ometry (MALDI-MS) were conducted to characterize the human genome-based rec ombinant proteins. Accurate mass values for the deleted forms of the myeloi d progenitor inhibitory factor chemokine (MPIF-1d23), and the keratinocyte growth factor (KGF-2d33) were measured as 8848.55 +/- 0.25 and 16,175.87 +/ - 0.89 Da, respectively. The mass accuracy of delayed ion extraction MALDI- MS measurements was within 20 ppm of the cDNA predicted value. Reduction an d alkylation of the chemokine showed the presence of six cysteine residues and three disulfide bonds, Additional confirmation of disulfide bonding amo ng the cysteine residues of the chemokine was demonstrated by identifying t he RP-HPLC separated tryptic and endoprotease Glu-C peptides, Three methion ine residues of the chemokine were identified by MALDI-MS of its cyanogen b romide (CNBr) cleavage products, The KGF-2d33 growth factor, however, lacke d the disulfide bonding between the two cysteine residues and contained two free sulfhydryl groups, Direct analysis of the growth factor CNBr digest s howed 7542.99, 4993.4, and 3107.7 Da peptides, which identified the methion ine residues. Peptide mapping mass spectrometry indicated that host-specifi c posttranslational modifications had not influenced the gene expression wo rk, (C) 1999 Academic Press.