Factors Influencing Phosphate Group Scrambling, and its Effect on Large-scale MS Based Phosphoproteomic Analysis

摘要

The magnitude of phosphate 'scrambling' has a strong dependence on the identity and number of the donor and acceptor residues. Serine has proven to be the best acceptor while the best donor is variable, depending on the properties of the peptides. The greater the number of donors and acceptors, the more likely 'scrambling' occurs. Increased rearrangement tends to occur with decreasing proton mobility. The longer the activation time (e.g., LTQ versus LTQ Velos), the higher the rearrangement ratio. Competing phosphate losses and rearrangement reactions both contribute to the abundance of –80 Da product ion. This results in ambiguity in using these product ion to localize the phosphate group. Phosphopeptides with increasing number of phosphorylated moieties and decreasing number of basic residues show a greater preference for lower charge states. This class of peptides require more attention when analyzing the results from large-scale phosphoproteome datasets due to the fact that they tend to have a higher propensity for 'scrambling'/ambiguous ions.