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

Abstract

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 P-mercaptoethanol.

Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13

“…PD mass spectra of the enzyme‐generated digests of rhIL‐13, and chromatographically isolated fractions thereof, were obtained on a BIOION 20 time‐of‐flight mass spectrometer. The instrument and the sample preparation procedures followed in the analysis of these samples have been described previously 24, 25…”

Applications of LC/MS in structure identifications of small molecules and proteins in drug discovery

“…This was confirmed by the 4‐Da mass shift of the measured MW of reduced rh‐GM‐CSF (treated with β‐mercaptoethanol) of 14 476 Da from nonreduced rh‐GM‐CSF of 14 472 Da. The MW information obtained from ESI‐MS spectrum has a remarkable mass accuracy (generally better than 0.01%) 60…”

LC/MS Analysis of Proteins and Peptides in Drug Discovery

“…It is an important physical parameter that can be used to confirm primary structure and identity of the protein, characterize post-translational modifications, and determine batch-to-batch reproducibility in the production of recombinant proteins. The mature protein sequence for human GM-CSF with four cysteine residues is shown in Table 19-1 [31]. Figure 19-3A displays the ESI-MS spectrum of rh-GM-CSF, containing a series of multiply-charged ions ranging from the 7+ to the 16+ charge state that correspond to molecular ions of the protein.…”

“…Interestingly, V 9 and V 12 peptides were not observed in the spectra despite their hydrophobic character based on primary structures. This signal suppression may arise from contributions of peptide's secondary or tertiary structure affecting its hydrophobic character [31]. To overcome the difficulty in detecting absent peptides, the mixture of digested V8 peptides was separated by HPLC and isolated fractions were analyzed by MS. All 13 V8 peptide fragments were revealed.…”

“…V 1 peptide was observed as V 1-2 at m/z 2302, while V 7 peptide was shown as part of V 7-8 at m/z 1824 due to incomplete cleavages. V 9 peptide was not only seen at m/z 3712 as expected, but was identified as V 9 -SS-V 12-13 (LYKQGLRGSLTKLKGPLTMMASHYK QHC 88 PPTPE-SS-NLKDFLLVIPFDC 121 WEPVQE, m/z 6017.6) and V 9 -SS-V [11][12][13] (LYKQGLRGSLTKLKGPLTMMASHYKQHC 88 PPTPE-SS- SFKENLKDFLLVIPFDC 121 WEPVQE, m/z 6508.7) [31]. These data clearly established another pairing of disulfide bond between Cys(88) and Cys (121).…”