Utility of reaction intermediate monitoring with photodissociation multi-stage (MS) time-of-flight mass spectrometry for mechanistic and structural studies: Phosphopeptides

In tandem mass spectra of phosphopeptides, intact sequence ions are often missing or appear weakly. Instead, dephosphorylated sequence ions appear prominently. In this work, we used photodissociation (PD) multi-stage (MSⁿ) time-of-flight mass spectrometry that can monitor reaction intermediates with lifetime as short as 100 ns to study the formation of dephosphorylated sequence ions such as y_n-H₃PO₄. y_n-H₃PO₄ was found to be formed mainly by H₃PO₄ loss from y_n. For doubly phosphorylated peptides, y_n seemed to lose H₃PO₄ stepwise and form y_n-H₃PO₄ and y_n-2H₃PO₄. Even when y_n was absent in PD-MS² spectrum, its m/z could be predicted from those of y_n-H₃PO₄ and/or y_n-2H₃PO₄. Complete sequence coverage was possible when the data from PD-MS² and PD-MS³ were combined, demonstrating the utility of transient ion detection by PD-MS³ for structure analysis.