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RATIONALE 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. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Protein post-translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the 'bottom-up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, 'top-down' MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top-down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post-translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27-42, 2013 [ABSTRACT FROM AUTHOR]