Selective detection of thiosulfate‐containing peptides using tandem mass spectrometry

Abstract

Incubation of proteins or peptides containing disulfide bonds (S-S) with sodium sulfite (Na(2)SO(3)) cleaves S-S bonds producing approximately equimolar amounts of free thiols (-SH) and thiosulfates (-S-SO(3)H), a process known as sulfitolysis. Proteins and peptides containing thiosulfates were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry (MS) and peptide mapping. The mass of the thiosulfate-containing peptide formed from oxidized insulin B chain was 3478.02 Da, 80 Da greater than the reduced peptide and corresponding precisely to addition of sulfur trioxide (SO(3)). Disulfide bond cleavage was also observed using RP-HPLC and MS after incubation of the intramolecular homodimer of mouse S100A8 (mass 20614 Da). The mass of HPLC-separated A8-SH was 10308 Da, and 10388 Da for A8-S-SO(3)H. Loss of SO(3) from multiply charged precursor ions was generally observed at elevated declustering potentials in the source region or within q(2) at relatively low collision energies (approximately 20 V). The characteristic loss of SO(3) at low collision energies preceded peptide backbone fragmentations at higher collision energies. Accurate mass measurement and charge-state discrimination, using a hybrid quadrupole time-of-flight mass spectrometer, allowed specific detection of thiosulfate-containing peptides. An information-dependent acquisition method, where the switch criterion was loss of m/z 79.9568, specifically identified 11 thiosulfate-containing peptides using nano-LC/MS from a tryptic digest of bovine serum albumin (BSA).

Decreased Immunoglobulin E (IgE) Binding to Cashew Allergens following Sodium Sulfite Treatment and Heating

“…We were unable to obtain confirmation of the precise location of the modified residue(s) within peptides because of the facile loss of the SO 3 modification prior to or during peptide fragmentation. A similar difficulty has been observed in attempts to specifically identify modified residues in insulin and BSA modified by sodium sulfite . We also observed evidence of a SO 3 modification within several Ana o 2 cysteine-containing peptides (Table ).…”

Top-down analysis of small plasma proteins using an LTQ-Orbitrap. Potential for mass spectrometry-based clinical assays for transthyretin and hemoglobin

“…The S -thiolated forms are quite stable and generally remain intact. On the other hand, the modification in S -sulfonated TTR is very labile; the S -sulfonate group on a cysteine residue is easily cleaved [38–39] and the released protein appears in the NSD mass spectrum as unmodified TTR. However although this post-translational modification is lost as a result of the activation method, the capability of the method to obtain the complete amino acid sequence information for variant characterization is not adversely affected.…”

Neural Network Modeling in Dithiothreitol Reduction and Ion Treatment of Recombinant Human Insulin Obtained from the Circular Dichroism (CD) Spectral Information

“…It is necessary for S-S bonds to be opened prior to final refolding. Such a process requires sulfur atoms to be converted into -SH functionality [4]. Despite the fact that the efficiency of protein isolation from inclusion bodies is high, such manipulations are crucial steps in the whole downstream process, especially for scale-up and manufacturing of recombinant protein from inclusion bodies.…”