Liquid chromatography/mass spectrometry analysis of branched fructans produced in vitro with ¹³C‐labeled substrates

Abstract

RATIONALE Fructans are carbohydrates predominantly based on fructose which are generally considered to be soluble dietary fibers with health‐promoting properties. It is known that the nutritional properties of fructans are affected by their structure. This study focused on structural determination of branched fructans, as the most important dietary fructans are branched graminan‐type fructans. METHODS Branched fructans were synthesized enzymatically by incubation of a heterologously expressed sucrose:fructan 6‐fructosyltransferase (6‐SFT) from Pachysandra terminalis with native or 13C‐labeled substrates. Liquid chromatography/mass spectrometry (LC/MS) was used for the structural identification of branched fructans. The MS2 fragmentation of these compounds is described for the first time. Analytes were charged by electrospray ionization in negative mode and a quadrupole mass analyzer was used for MS2 analysis. RESULTS The MS2 fragmentation patterns of branched and linear fructans were shown to differ and distinctive ion formation allowed differentiation between all branched fructan isomers formed. P. terminalis 6‐SFT preferred extending the existing fructan branch rather than creating a new branch. CONCLUSIONS The MS2 fragmentation patterns described in the current paper now allow rapid screening of large sample sets for the presence of branched, graminan‐type fructans. Furthermore, the data enables the characterization of fructan‐metabolizing enzymes by identification of the fructan structures produced by in vitro reactions as described here for P. terminalis 6‐SFT. Copyright © 2014 John Wiley & Sons, Ltd.

LC-MS analysis reveals the presence of graminan- and neo-type fructans in wheat grains

“…Sucrose, 6-kestotriose (6-K), 1-kestotriose (1-K), 6,6-kestotetraose (6,6-KT), 1&6-kestotetraose (1&6-KT), 1,1-kestotetraose (1,1-KT), 1&6,6-kestopentaose (1&6,6-KP), 6&1,6-kestopentaose (6&1,6-KP), 6,1&1-kestopentaose (6,1&1-KP) and 1&6,6,6-kestohexaose (1&6,6,6-KH) are depicted. The presence of 6&1,6-kestopentaose and 6,1&1-kestopentaose could not be confirmed by injection of an (enzymatically synthesized) fructan standard with known structure (Verspreet et al, 2014b) and their formation is represented by a dashed reaction arrow.…”

“…Graminan-type fructans were synthesized in vitro by a Pachysandra terminalis 6-SFT (sucrose:fructan 6-fructosyltransferase) (Van den Ende et al, 2011) with native or 13 C labeled substrates. Comparison of the MS 2 spectra of native and labeled graminans allowed their structural identification (Verspreet et al, 2014b). Developing wheat grains sampled at 0, 2, 7, 14, 20, 28 and 42 days after anthesis (DAA) were ground to a wheat whole meal and lyophilized as described before (Verspreet et al, 2013a).…”

“…Standard injection and MS 2 analysis (Harrison et al, 2012;Verspreet et al, 2014b) demonstrated that 1-K and 6-K were the only fructan trisaccharides present at anthesis whereas 1&6-kestotetraose (1&6-KT) and 1,1-KT were the major DP 4 fructans (Fig. 1A).…”

“…Data were collected in centroid mode and analyzed with Xcalibur 3.0 software. Carbohydrates were identified by injection of commercial available standards and enzymatically synthesized fructan standards (Verspreet et al, 2014b) and by comparison of the MS 2 spectra with earlier published data (Harrison, 2012;Harrison et al, 2012). The nomenclature of Domon and Costello (1988) was used for the designation of the fructan fragments formed.…”

“…Fragments containing the glucose end are labeled as A i , B i and C i , with i representing the number of the glycosidic bond cleaved, counting from the glucose end. Fragments with a terminal fructose end are designated as X j , Y j and Z j and the j index counting starts from the fructose end (Verspreet et al, 2014b). The letters A and X are used to designate fragments formed by cross ring cleavage.…”