High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear...

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS²) with electron transfer dissociation (ETD) has proven to be a promising method for phosphoproteomic applications owing to its ability to determine phosphorylation sites on proteins. However, low precursor charge states hinder the ability to obtain useful information through peptide sequencing by ETD, and the presence of acidic phosphate groups contributes to a low charge state of peptide ions. In the present report, we used a dinuclear zinc complex, (Zn₂L)³⁺ (L = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol) for electrospray ionization (ESI), followed by ETD-MS² analysis. Since (Zn₂L)³⁺ selectively bound to phosphopeptide with addition of a positive charge per phosphate group, the use of (Zn₂L)³⁺ for ESI improved the ionization yield of phosphopeptides in phosphoprotein digest. Additionally, an increase in the charge state of phosphopeptides were observed by addition of (Zn₂L)³⁺, facilitating phosphopeptide sequencing by ETD-MS². Since the binding between (Zn₂L)³⁺ and the phosphate group was retained during the ETD process, a comparison between the ETD mass spectra obtained using two dinuclear zinc complex derivatives containing different zinc isotopes, namely (⁶⁴Zn₂L)³⁺ and (⁶⁸Zn₂L)³⁺, provided information about the number of phosphate groups in each fragment ion, allowing the phosphorylation site to be unambiguously determined. The details of the fragmentation processes of the (Zn₂L)³⁺-phosphopeptide complex were investigated using a density functional theory calculation. As in the case of protonated peptides, ETD induced peptide backbone dissociation in the (Zn₂L)³⁺-phosphopeptide complex proceeded through an aminoketyl radical intermediate.