Residue-Specific Gas Phase Peptide Fragmentation
Conventional approaches to gas phase peptide fragmentation are collision induced dissociation (CID), infrared multiphoton photodissociation (IRMPD), electron capture dissociation (ECD), and more recently, electron transfer dissociation (ETD). Generally, both of these fragmentation processes occur randomly along the peptide backbone with limited exceptions (such as the proline/aspartic acid effect [CID], and disulfides [ECD, ETD]). These techniques often generate large data sets, which must be checked against an even larger database of possible fragments (predicted in silico) to identify a protein.
We propose an alternative approach to peptide fragmentation by developing techniques that are selective for specific protein residues. In theory, fragmentation thus obtained would be subject to certain “rules” that would limit the number of fragmentation sites possible (analogous to an enzymatic digest) thereby reducing computational cost.
Chemically accessible tyrosines on a protein are modified with iodine to form iodotyrosine (Figure 1a). These non-native carbon-iodide bonds are photolabile and undergo direct dissociation to generate a protein radical, localized to the modified tyrosine (Figure 1b). CID of the nascent protein radical produces selective dissociation at tyrosine (Figure 1c).
The posterior plate of our LTQ linear ion trap mass spectrometer is modified with an optically transparent quartz window to introduce fourth harmonic (266 nm) laser pulses from a Nd:YAG laser (Figure 2). Laser shots are synchronized to the beginning of the activation step of a typical MS2 experiment.