Structural distinction among inositol phosphate isomers using high-energy and low-energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.

Abstract

Electrospray (ESI) collisional-activated dissociation (CAD) tandem mass spectrometric methods for the structural characterization of inositol phosphates (InsPs) using both quadrupole and sector mass spectrometers are described. Under low-energy CAD, the [M + H](+) ions of the positional isomers of inositol phosphates, including inositol mono-, bis- and trisphosphates, yield distinguishable product-ion spectra, which are readily applicable for isomer differentiation. In contrast, the product-ion spectra arising from high-energy CAD (2 keV collision energy, floating at 50%) tandem sector mass spectrometry are less applicable for isomer identification. The differences in the product-ion spectrum profiles among the aforementioned InsP isomers become more substantial and differentiation of positional isomers can be achieved when the collison energy is reduced to 1 keV (floating at 75%). These results demonstrate that the applied collision energies play a pivotal role in the fragmentations upon CAD. The product-ion spectra are similar among the positional isomers of inositol tetrakisphosphates and of inositol pentakisphosphates. Thus, isomeric distinction for these two inositol polyphosphate classes could not be established by the tandem mass spectrometric methods that have achieved such distinctions for the less highly phosphorylated inositol phosphate classes. Under both high- and low-energy CAD, the protonated molecular species of all InsPs undergo similar fragmentation pathways, which are dominated by the consecutive losses of H(2)O, HPO(3) and H(3)PO(4).