Characterization of disulfide bonds by planned digestion and tandem mass spectrometry

Abstract

The identification of disulfide bonds provides critical information regarding the structure and function of a protein and is a key aspect in understanding signaling cascades in biological systems. Recent proteomic approaches using digestion enzymes have facilitated the characterization of disulfide-bonds and/or oxidized products from cysteine residues, although these methods have limitations in the application of MS/MS. For example, protein digestion to obtain the native form of disulfide bonds results in short lengths of amino acids, which can cause ambiguous MS/MS analysis due to false positive identifications. In this study we propose a new approach, termed planned digestion, to obtain sufficient amino acid lengths after cleavage for proteomic approaches. Application of the DBond software to planned digestion of specific proteins accurately identified disulfide-linked peptides. RNase A was used as a model protein in this study because the disulfide bonds of this protein have been well characterized. Application of this approach to peptides digested with Asp-N/C (chemical digestion) and trypsin under acid hydrolysis conditions identified the four native disulfide bonds of RNase A. Missed cleavages introduced by trypsin treatment for only 3 hours generated sufficient lengths of amino acids for identification of the disulfide bonds. Analysis using MS/MS successfully showed additional fragmentation patterns that are cleavage products of S-S and C-S bonds of disulfide-linkage peptides. These fragmentation patterns generate thioaldehydes, persulfide, and dehydroalanine. This approach of planned digestion with missed cleavages using the DBond algorithm could be applied to other proteins to determine their disulfide linkage and the oxidation patterns of cysteine residues.

Figure 1

Types of disulfide-linked peptides. Peptides that contain a single disulfide bond are called simple forms; other forms are referred to as complex. a) A simple form of inter-disulfide–linked peptide is termed “desirable” when the lengths of its component peptides are longer than five amino acids and the peptide mass is less than 5000 Da. b) Intra-peptide bonds and c) inter- and intra-peptide bonds can be generated from the oxidation of cysteine thiols.

Figure 2

Known disulfide bonds (linked by red line) in the RNase A protein sequence. Based on the known disulfide bonds, four disulfide-linked products were digested in silico using a) trypsin; b) chymotrypsin + trypsin; c) Asp-N/C + LysC; d) Asp-N/C + trypsin.

Figure 3

Annotated MS/MS spectra of (a) ‘SSTSAASSSNYCNQMMK–CR’ and (b) ‘SSTSAASSSNYCNQMMK–CRETGSSK’ disulfide-linked peptides. Matched peaks are shown in red. Ion annotations used: ‘++’ and ‘3+’, doubly and triply charged fragment ions, respectively; ‘P^A’, one strand (top) of a dipeptide; ‘p_b’, the other strand (bottom) of a dipeptide; uppercase letters, fragment ions from peptide P^A; lowercase letters, fragment ions from peptide p_b; ‘+32’ and ‘−34’, persulfide and dehydroalanine ions, respectively, formed by C–S bond cleavage reactions. In particular, fragment ions from peptide p_b are underlined and are observed only for ‘CRETGSSK’.

Figure 4

Additional fragmentations of C–S and S-S bond cleavages. a) Fragmentation patterns for persulfide and dehydroalanine fragment ions from C–S bond cleavage reactions, and cysteine and thioaldehyde fragment ions from S–S bond cleavage reactions. b) Observed persulfide (+32 Da) and dehydroalanine (−34 Da) ions are circled.