Phosphate Transfer in Activated Protein Complexes Reveals Interaction Sites

Abstract

For many proteins, phosphorylation regulates their interaction with other biomolecules. Herein, we describe an unexpected phenomenon whereby phosphate groups are transferred non-enzymatically from one interaction partner to the other within a binding interface upon activation in the gas phase. Providing that a high affinity exists between the donor and acceptor sites, this phosphate transfer is very efficient and the phosphate groups only ligate to sites in proximity to the binding region. Consequently, such phosphate-transfer reactions may define with high precision the binding site between a phosphoprotein and its binding partner, as well as reveal that the binding site in this system is retained in the phase transfer from solution to the gas phase.

Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

“…However, with improvements in instrumentation (e.g., nano-electrospray ionization), as well as other advances in the fields of native mass spectrometry (native MS) and ion mobility-mass spectrometry (IM-MS), data supporting a more gentle gas-phase transition started to accumulate [ 23 , 24 ]. Furthermore, nonergodic fragmentation techniques that preferentially preserve noncovalent interactions (e.g., electron-transfer dissociation) revealed similarities between native-like structures of proteins observed in the solution and in the gas phase [ 25 – 28 ]. Taken together, accumulating all the evidence, it has become clear that it is possible to retain partially solution-phase properties of proteins and protein complexes in the gas phase, although it remains system dependent.…”

Phosphopeptide Fragmentation and Site Localization by Mass Spectrometry: An Update

“…UV-PD has been exploited to phosphopeptides to identify and site-localize the phosphate moiety. Early results indicate that UV-PD is compatible with the high-throughput identification of phosphopeptides while the propensity of the phosphate group to remain bound to the peptide during fragmentation increases when compared to HCD. − Top-down and middle-down phosphoproteomics approaches, which have greatly benefited from recent technological advances in alternative fragmentation techniques may improve the identification and site localization of protein phosphorylations and be ideally suited to not only map multiple modifications on a peptide or protein at once but also determine phospho-isoform abundance, reaction kinetics and to study functional cross-talk between post-translational modifications. ,− …”