Determination of phosphate position in hexose monosaccharides using an FTICR mass spectrometer: ion/molecule reactions, labeling studies, and dissociation mechanisms

Determination of phosphate position in carbohydrates using mass spectrometry is difficult due to the low energy loss of the phosphate either as a neutral or as an ion in MS/MS experiments. A possible solution to this problem is proposed in this work, whereby we use ion/molecule reactions in combination with tandem mass spectrometry to determine the site of phosphorylation on phosphorylated monosaccharides. Singly charged negative ions from phosphorylated monosaccharides are reacted with trimethyl borate in an FTICR MS analyzer cell to produce ion/molecule reaction products with the loss of a neutral methanol molecule. This reaction product likely involves a covalent bond between one of the phosphate oxygen atoms and boron. Derivatization of the phosphate in this manner allows stabilization of the phosphate group under SORI-CID conditions, allowing generation of ions characteristic of the phosphate linkage. Ion structures and dissociation mechanisms explaining these results are presented and discussed. The mechanistic studies suggest that the extra degrees of freedom provided by the 6-linked phosphate allows formation of diagnostic ions in the 6-linked case that are not formed from the 1-linked isomer. The dissociation of the ion/molecule reaction products using infrared multi-photon dissociation (IRMPD) as an activation method was also investigated. While SORI-CID and IRMPD activation yield similar dissociation patterns, the characteristic differences in the product ion spectra between the monosaccharides phosphorylated in the 1- and 6-positions are not observed using IRMPD.