An On-Target Performic Acid Oxidation Method Suitable for Disulfide Bond Elucidation Using Capillary Electrophoresis - Mass Spectrometry

Abstract

Disulfide bonds play important roles in establishing and stabilizing three-dimensional protein structure, and mass spectrometry (MS) has become the primary detection method to decipher their biological and pathological roles. Several experimental methods before or after MS detection have been developed to aid in disulfide bond assignment, such as tandem MS followed by database searching or modification of the disulfide bond via chemical reduction or oxidation. Despite these technological advancements, the detection and proper assignment of disulfide bonds have remained experimentally difficult. Therefore, we have developed an alternative method for disulfide bond elucidation using capillary electrophoresis-mass spectrometry (CE-MS) combined with an on-target performic acid oxidation method for matrix assisted laser desorption/ionization (MALDI) deposited samples. An information rich CE-MS method that results in distinct charge-state trends observed in two-dimensional plots of log(mu eff) versus log (MW) was developed to enhance the confidence of peptide and protein identifications. The charge-state trends provide information about the number of basic amino acid residues present within each peptide. This information can be used to develop methods to screen for posttranslationally modified peptides (e.g., phosphorylation, disulfide bonds, etc.). In the case of disulfide bonds, the highly charged peptides (i.e., 3, 4 or greater charge states) have a high probability of being disulfide-linked peptides, owing to charge contribution of both peptides forming the disulfide bridged peptide. However, intra-linked disulfide bridged peptides can also be present at lower charge states. Therefore, a chemically selective method to rapidly locate disulfide-linked peptides that have been separated by CE-MS must be developed. An on-target performic acid oxidation method was developed to provide the chemical selectivity towards disulfide bonds, i.e., converting the cystine bond to form two peptides modified with a cysteic acid (SO3H) side chain. The on-target oxidation method offers (i) no post-oxidation sample cleanup, (ii) improved throughput over solution-phase oxidation methods, and (iii) easily adapted to CE separations coupled offline with MALDI-MS. The evaluation of the on-target oxidation experimental parameters, the fragmentation behavior of cysteic acid-containing peptides and an alternative method for disulfide bond elucidation, using CE-MS combined with the ontarget oxidation method, are discussed within.