Determination of Peptide and Protein Disulfide Linkages by MALDI Mass Spectrometry

Abstract

The disulfide bond is one of the most common post-translational modifications in proteins, of which determination is essential to the comprehensive understanding of protein structures. Disulfide bond analysis has undergone great improvement due to the development of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), especially in terms of speed and sensitivity. In general, the characterization of disulfide-containing peptides is achieved by the reduction of disulfide bonds followed by alkylation. In this review we focus on the analysis of disulfide-containing proteins/peptides by some unique methods in MALDI MS. The MALDI in-source decay (ISD) of disulfide bonds and adducts of the matrix and sulfhydryl-containing peptide are discussed in detail. The mechanism of each method is discussed so as to help the reader gain greater insight into it, and examples of its application are also presented. The goal of this review is to provide an understanding of these techniques for analysis of disulfide-linked proteins/peptides in MALDI MS.

Molecular Characterization and Functional Analysis of the Nattectin-like Toxin from the Venomous Fish Thalassophryne maculosa

“…We can observe in Figure 1C that the treatment generated proteins with masses of 15,484.8 and 15,982.8. By the difference of the masses of the pure samples and the reduced/alkylated samples, we prove the presence of 6 cysteine residues in each of the proteins according to Yang, Liu, and Liu [18].…”

“…We can observe in Figure 1C that the treatment generated proteins with masses of 15,484.8 and 15,982.8. By the difference of the masses of the pure samples and the reduced/alkylated samples, we prove the presence of 6 cysteine residues in each of the proteins according to Yang, Liu, and Liu [18]. The raw venom of Thalassophryne maculosa was submitted to a sequence of fractionation in a high-pressure liquid chromatography (HPLC) system using a C18 followed by C8 reversed phase HPLC columns, according to the scheme in (A) (top left).…”

Magnetic silica nanoparticles for use in matrix-assisted laser desorption ionization mass spectrometry of labile biomolecules such as oligosaccharides, amino acids, peptides and nucleosides

“…[6,7]. Typically an organic matrix is used to facilitate the ionization of macromolecules, but detection of small molecules (<700 Da) has been a challenge in MALDI MS due to interference from conventional matrices in the low mass region [8][9][10].…”

Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay

“…The presence of disulfide bonds was for example probed by collision-induced dissociation (CID) [22] and the disulfide connectivties were determined using electron transfer dissociation (ETD) [24,25]. MALDI in-source decay (ISD) reactivity of disulfide bonds has also been investigated on various peptides [26][27][28][29][30][31][32]. These studies pointed out the ability of 1,5-diaminonaphtalene (1,5-DAN) matrix to reduce disulfide bonds (addition of two radical hydrogens on the sulfur atoms) and the possibility to determine the number of S-S bonds in a given peptide (from 0 to 3).…”