Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology

Abstract

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a β-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Keywords: cardiotoxin; disulfide bond analysis; folding isomer; mass spectrometry.

Figure 1

Characterization of synthetic cardiotoxin polypeptide (syn-CTX A3) using liquid chromatography coupled with the ultraviolet detector and electrospray ionization mass spectrometry (LC–UV/ESI–MS). (A) One major peak was observed in the LC chromatogram of synthetic polypeptide under the detection wavelength of UV 214 nm. (B) The ESI–MS spectrum of synthetic polypeptide showed multiple peak profiles with different charge states: m/z 676.0 (+10), m/z 750.8 (+9), m/z 844.4 (+8), m/z 964.8 (+7), and m/z 1125.2 (+6).

Figure 2

Comparison of MS/MS spectrum of disulfide linked peptides derived from the folding intermediates (A,B) and native CTX A3 protein (C,D). The MS/MS spectra were acquired from the precursor ion of (A) m/z 565.32 (+2), (B) m/z 701.32 (+4), (C) m/z 565.32 (+2), and (D) m/z 701.62 (+4), respectively. Asterisks (*) indicate the dimethyl labeling sites in the peptides. The CID fragments of the a1/y ion signals are annotated in each spectrum. N-amide represents the amidated asparagine (N) residues in the C-terminals of the peptides.

Figure 3

Time course relative peak area graph of (A) disulfide-linked peptides with either single or triple native cysteine pairings, and (B) disulfide-linked peptides with non-native disulfide bonds. Each relative peak area graph of disulfide-linked peptide was annotated with their corresponding cysteine pairings listed in Table 1.

Figure 4

Analysis of secondary structural analysis of fractionated folding intermediate using CD specrometer. (A) HPLC chromatogram of folding intermediate (48hr). The sample was separated with reverse phase column, and eight fractions, A-H, were collected for further CD analysis. (B) CD measurement of HPLC fraction A to H. The acquired spectra were characterized and combined for comparative analysis.

Figure 5

In vitro cell-based cytotoxicity analysis. The sigmoid curve of cell viability was acquired by treating a constant amount of HL-60 cells with incubated with various concentrations of (A) folded syn-CTXA3 with native disulfide bonds, or (B) folding isomers with non-native disulfide bonds. The assay was performed in triplicate. The dashed line indicates the sample concentration that caused death of half of the cells. The dashed line indicates the toxin concentration that caused death of half of the cells.

Figure 6

(A) The Ab titer (log value) of mouse serum against the native CTX A3 toxin. Blank represents the pre-immune mouse serum for comparative purposes. (B) In-vitro cell-based neutralization assay. The sigmoid curve of cell viability was obtained by treating HL-60 cells with a mixture of varying doses of native CTX A3 toxin together with a constant amount of antisera. The cells incubated with native CTX A3 toxin alone were utilized as controls (•) in the assay. The neutralizing potency of mouse antisera against cytotoxicity was calculated by the equation described in the experimental section.