Selective biochemical modification of functional residues in recombinant human macrophage colony-stimulating factor beta (rhM-CSF beta): identification by mass spectrometry.
A rapid method combining classical chemical modification with mass spectrometry was developed to identify amino acids in the recombinant human macrophage colony-stimulating factor (rhM-CSF) protein of potential import to the ligand-receptor interaction. Diethyl pyrocarbonate modification of rhM-CSF beta (under nondenaturing conditions) results in a time- and concentration-dependent loss in receptor binding and biological activity. Peptide mapping of the reaction products by mass spectrometry showed that, with low DEP:M-CSF ratios (< 50:1), there was selective modification of histidine residues, whereas at higher ratios (> 50:1), Tyr and Lys residues were also modified. The loss in rhM-CSF beta activity was directly correlated with the extent of carbethoxylation of His9 and His15, as determined by matrix-assisted laser desorption/ionization mass spectrometric molecular weight determinations (MALDIMS). For these residues mono-modification was observed. By contrast, C-terminal histidine residues His176 and His210 showed bis-modifications, the extent of which had no correlation to losses in biological activity. These data suggest the importance of residues in the A-helix (His9 and His15) to ligand-receptor binding.
Glocker
M O
MO
Faculty of Chemistry, University Konstanz, Germany.
Kalkum
M
M
Yamamoto
R
R
Schreurs
J
J
D000595:Amino Acid Sequence; D004047:Diethyl Pyrocarbonate; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor; D014357:Trypsin; D004047:Diethyl Pyrocarbonate