Disulfide linkages in the in vitro refolded intermediates of recombinant human macrophage-colony-stimulating factor: analysis of the sulfhydryl alkylation of free cysteine residues by fast-atom bombardment mass spectrometry.

Abstract

Fast-atom bombardment mass spectrometry was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophage-colony-stimulating factor. The content of iodoacetamide-alkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion high-performance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys157 and Cys159 and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.

Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone

“…The second approach has been developed for the situation where proteolytic cleavage results in individual peptides that contain more than one cysteinyl residue. The absence or presence of disulfide bonds in these peptides is detected by first alkylating free thiols in the intact protein followed by proteolytic cleavage and subsequent detection of the peptide masses (4,(12)(13)(14). Thiol-containing peptides are found alkylated and are clearly distinguished by the corresponding mass shifts from unmodified and, thus, disulfide bond-carrying peptides.…”

Early risk prognosis of free-flap transplant failure by quantitation of the macrophage colony-stimulating factor in patient plasma using 2-dimensional liquid-chromatography multiple reaction monitoring-mass spectrometry

“…M-CSF is a hematopoietic growth factor [44,45] that is involved in proliferation, differentiation, and survival of the monocyte/macrophage lineage. [46–48] A variety of cell types including macrophages, endothelial cells, fibroblasts, lymphocytes, and bone marrow-derived stromal cells produce M-CSF.…”

Selective Bridging of Bis-Cysteinyl Residues by Arsonous Acid Derivatives as an Approach to the Characterization of Protein Tertiary Structures and Folding Pathways by Mass Spectrometry

“…Reduced OT was modified at different concentrations (15,30,45,60, and 120 M) with 45 M MEL solutions in 50 mM NaH 2 PO 4 (pH 8), containing 2.5 mM EDTA, and in 50 mM NH 4 OAc (pH 4), respectively. Final volume was adjusted to 1 ml for UV spectroscopic studies.…”

“…13 and 14 for reviews on this subject). The correct formation of disulfide bonds is crucial for folding into the "native" three-dimensional protein structure (15). Hence, the identification of bissulfhydryl groups in reduced proteins which are suitably spaced to form disulfide bridges is important for the characterization of tertiary structures.…”