Selective Biochemical Modification of Functional Residues in Recombinant Human Macrophage Colony-Stimulating Factor β (rhM-CSF β):  Identification by Mass Spectrometry

• Published in: Biochemistry (Easton) Vol. 35; no. 46; pp. 14625 - 14633
• Main Authors: Glocker, M. O, Kalkum, M, Yamamoto, R, Schreurs, J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.11.1996
• Subjects: Amino Acid Sequence; Diethyl Pyrocarbonate; Granulocyte-Macrophage Colony-Stimulating Factor - chemistry; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Humans; Mass Spectrometry - methods; Models, Molecular; Molecular Sequence Data; Molecular Weight; Receptor, Macrophage Colony-Stimulating Factor - metabolism; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961199o

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 β (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 β 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 (MALDI-MS). 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.