Using mass spectrometry with electron transfer dissociation, a 2010 study by a team in Boston confirmed the location and status of the unpaired cysteine in recombinant-tissue plasminogen activator (rt-PA) and mapped all of its disulfide bonds. Clinicians administer rt-PA to halt blood clotting caused by heart attacks, ischemic strokes, and pulmonary embolisms. An enzyme, tissue plasminogen, prompts a chemical cascade leading to fibrinolysis — the destruction of fibrinogen, a primary component in blood clotting. When applied to ischemic stroke early on, rt-PA significantly improves patient outcome and reduces subsequent ischemic events.1 Currently, the U.S. Federal Drug Administration has approved three versions of recombinant plasminogen activator, including alteplase, reteplase, and tenecteplase. One of the mysteries that chemical biologists have been working on is the location of an unpaired cysteine in rt-PA.
In their neutral state, cysteines are paired through disulfide bonds. This pairing is integral to protein folding. Unpaired cysteines can be reactive in redox reactions, leading to covalent association, enzyme catalysis, or disulfide scrambling. Researchers have also established that the antioxidant glutathione can block unpaired cysteines. Produced by cells, glutathione neutralizes free radicals and reactive oxygen compounds. The reaction of glutathione with cysteine can be a mechanism for pathway signaling, and researchers often use glutathione to locate potential targets for pharmaceutical intervention.1
Up until the 2010 study, no direct evidence had been presented for rt-PA’s complete structure. Using a new method, combining liquid chromatography mass spectrometry (LC-MS) with electron transfer dissociation and collision-induced dissociation, the Boston team characterized all 17 disulfide bonds. They also determined that, when unpaired, the resulting free sulfhydryl group is reactive and that pairing with either glutathione or cysteine may be protective. They speculate that under oxidative or ischemic stress, either glutathione or cysteine might trigger the pairing molecule to free itself for protein activation, as found for other proteins in biological systems. Electron transfer dissociation made it possible for the team to identify an O-linked fucose, which likely masked the unpaired status of the cysteine in other experiments, making it difficult to find.1
The identification of the location and status of the unpaired cysteine furthers the study of the activation of rt-PA by hypoxia for ischemic stroke patients and oxidative stress due to heart attack.1
1. Wu, S.L., et al. (2010) ‘Identification of the unpaired cysteine status and complete mapping the 17 disulfides of recombinant tissue plasminogen activator using LC-MS with ETD/CID‘, Analytical Chemistry, 82 (12), (pp. 5296-5303)