Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS

Abstract

Through the use of ion/molecule reactions and tandem mass spectrometry, phosphate position is assigned in both phosphorylated monosaccharides and oligosaccharides. In previous work [1, 2] phosphate moieties of monosaccharides were stabilized under collisional activation, by first derivatizing the deprotonated monosaccharide with trimethyl borate through an ion/molecule reaction, and the phosphate position determined through marker ions generated in tandem mass spectra. In this work, the methodology is extended to larger phosphorylated oligomers employing chlorotrimethylsilane (TMSCl) as the ion/molecule reagent. Phosphorylated monosaccharides were first investigated to determine diagnostic ions for phosphate linkage in monomeric standards. It was observed that the diagnostic ions showed both linkage and some monosaccharide stereochemical information. Furthermore, it was observed that TMS addition stabilized the phosphate moiety under collisionally activated conditions. Upon identification of the diagnostic ions, the methodology was applied to lactose-1-phosphate. It was found that TMSCl, stabilized the phosphate moiety upon collisional activation, and furthermore, the phosphate linkage could be determined through tandem mass spectrometric analysis. As a further extrapolation to biologically relevant problems, the methodology was applied to a lipophosphoglycan analog from the protozoan parasite Leishmania. This sample contains bridging phosphates which were converted to terminal phosphates through collision induced dissociation. The sample was then analyzed in the same manner as lactose-1-phosphate, yielding phosphate linkage information and stereochemical information. This study showed that, using the developed methodology, phosphate linkage can be determined from both monosaccharides and larger oligosaccharides; furthermore it is applicable to samples in which the phosphates are either terminating or bridging. (J Am Soc Mass Spectrom 2003, 14, 323-331)

Selective Detection of Sugar Phosphates by Capillary Electrophoresis/Mass Spectrometry and Its Application to an Engineered E. coli Host

“…The main challenge in sugar phosphate analysis by MS alone is that sugar phosphate isomers have the same m/z values and display similar MS-MS fragmentation patterns. [10] Therefore, in order to differentiate between sugar phosphate isomers, it would be necessary to introduce a separation step prior to their analysis by mass spectrometry. As mentioned earlier, CE is a particularly useful separation technique in this study, as it is suited to the separation of charged and polar molecules and has demonstrated the capability to separate regioisomers such as a-d-glucose-1-phosphate and d-glucose-6-phosphate on the basis of subtle differences in their pK a values.…”

“…[1][2][3] Sugar phosphates are inherently polar compounds and possess a phosphate functional group, and the use of CE-ESMS in the negative mode would thus be a suitable choice of analy-A C H T U N G T R E N N U N G tical technique. [10,11] CE offers several advantages over other commonly used separation techniques, such as liquid chromatography (LC), in this context because it: 1) is particularly suited towards analyzing highly polar ionic molecules; 2) demonstrates better separation efficiency, which is ideal for resolving complex biological cell extracts and; 3) requires only small amounts of sample (in the low nL range). [12][13][14][15][16][17] In addition, the determination of the anomeric configurations of sugar phosphates by mass spectrometry can pose a challenge because regioisomers have identical m/z values.…”

“…The use of CE with indirect UV has permitted the analysis of regioisomers, [18] but with regard to mass spectrometry, the differentiation of anomeric isomers has generally required tandem MS [11] or derivatization of the isomers with an ion/molecule reagent to produce diagnostic ions. [10] Here, we have evaluated the selectivity of this current CE-ESMS method as a tool to separate and identify A C H T U N G T R E N N U N G regioisomers without the need for sample derivatization.…”

Anomeric information obtained from a series of synthetic trisaccharides using energy resolved mass spectra

“…Collision induced dissociation (CID), especially one carried out on an ion trap mass spectrometer (ITMS), is extremely important because it allows one to obtain detailed information of the precursor ion in focus through iterative ion‐molecule reactions at multiple CID stages (MS n ) 7–11. Some reports have described differences in the intensities and more recently energy‐resolved mass spectra (ERMS) of respective pairs of fragment ions from a pair of synthetic and naturally occurring oligosaccharides 12–18. However, such a comparison can only be made when a set of isomers is available.…”

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers: An FT-ICR approach

“…Until recently, very little information existed for determining phosphorylation sites in carbohydrates because dissociation of underivatized phosphorylated oligosaccharides generates mainly phosphate cleavages [13]. Recently, we developed a method to determine the phosphate position of phosphorylated carbohydrates by using ion/molecule reactions and FT-ICR mass spectrometry [14,15]. Gas-phase reactions between phosphorylated hexose ions (HexXP, where X ϭ 1 or 6 indicating the linkage of the phosphate to the monosaccharide) and trimethyl borate (TMB) or trimethyl chloro-silane (TMSCl) result in distinguishable mass spectra.…”