Probing the exposure of the phosphate group in modified amino acids and peptides by ion-molecule reactions with triethoxyborane in Fourier transform ion cyclotron resonance mass spectrometry.
BACKGROUND
Intramolecular hydrogen bonds between a phosphate group and charged residues play a crucial role in the chemistry of phosphorylated peptides, driving the species to specific conformations and affecting the exposure of the phosphate moiety. The nature and extent of these interactions can be investigated by measuring the reactivity of phosphate groups toward selected substrates in the gas phase.


METHODS
We used Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS) to perform a systematic study on the gas-phase ionic reactivity of phosphorylated amino acids and peptides with triethoxyborane (TEB). Ions of interest were generated by electrospray ionization (ESI), isolated in the cell of the FT-ICR mass spectrometer, and allowed to react with a stationary pressure of TEB. The temporal evolution of the reaction was monitored and thermal rate constants were derived. The structure of the ionic products was confirmed by Collision-Induced Dissociation (CID) tandem mass spectrometry (MS/MS).


RESULTS
TEB was found to react with the phosphate of protonated phosphorylated amino acids and peptides by an addition-elimination pathway. The kinetic efficiency of the reaction showed a positive correlation with the charge state of the reagent ion, suggesting the existence of charge-state-dependent exposure of the phosphate groups towards the incoming neutral during the reaction. Isomeric phosphorylated peptides, only differing for the position of the modified serine residue, showed markedly different kinetic efficiencies.


CONCLUSIONS
The ability of a phosphorylated species to react with TEB depends on the ease of access to the phosphate moiety in the corresponding gaseous ion. Measuring the kinetic efficiency of such reactions can represent a valuable tool to explore the accessibility of phosphate groups in biomolecules.
Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester, M1 7DN, UK; Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
Francesco
F
Lanucara
Simonetta
S
Fornarini
Claire E
CE
Eyers
Maria Elisa
ME
Crestoni
D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D001880:Boranes / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D010710:Phosphates / Q000032:analysis / Q000737:chemistry*; D011522:Protons
D000596:Amino Acids; D001880:Boranes; D010455:Peptides; D010710:Phosphates; D011522:Protons