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-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.
Tsarbopoulos
A
A
Shering-Plough Research Institute, Kenilworth, New Jersey 07033.
Pramanik
B N
BN
Labdon
J E
JE
Reichert
P
P
Gitlin
G
G
Patel
S
S
Sardana
V
V
Nagabhushan
T L
TL
Trotta
P P
PP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin