Analysis of Peptide Photofragmentation for Single-Molecule Protein Sequencing

We analyze the feasibility of using light to fragment a peptide into its constituent amino acids before identifying them by mass spectrometry (MS) for the purpose of single-molecule protein sequencing. Laser power considerations strongly favor photofragmenting peptides in solution before they leave the ion source rather than in the gas phase. Ultaviolet (UV) wavelengths near 200 nm are weakly absorbed in water, and a single photon can selectively cleave a peptide bond that links amino acids together. These properties make UV photofragmentation more promising than methods based on infrared or x-ray light. We develop a simple model of the probability of liberating an amino acid intact by cleaving the peptide bonds on either side of it before the light damages the amino acid itself. We predict 193 nm light can liberate many amino acids with probabilities ranging from 0.65-0.92; however, the aromatic amino acids and histidine, me- thionine, arginine, and lysine, which are relatively susceptible to photodamage, would be liberated intact with a probability in the range 0.004-0.330. These findings suggest that UV photofragmentation could reveal a significant amount of a single protein’s sequence information to a mass spectrometer.