ISOLATION AND CHARACTERIZATION OF A RESISTANT CORE PEPTIDE OF RECOMBINANT HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) -CONFIRMATION OF THE GM-CSF AMINO-ACID-SEQUENCE BY MASS-SPECTROMETRY

A trypsin-resistant core peptide of recombinant human granulocyte-macr ophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analy sis. This analysis provided successful detection of the high-mass disu lfide-linked core peptide as well as information confirming the existe nce of disulfide pairing. Similarly, LSI mass spectrometric analysis o f the peptide fragments isolated chromatographically from a Staphyloco ccus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulf ide bonds between cysteine residues 54-96 and 88-121. Electrospray ion ization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bo nds in the protein molecule by comparative analysis of the protein bef ore and after reduction with beta-mercaptoethanol.