A Novel Method for Identification and Quantification of Sulfated Flavonoids in Plants by Neutral Loss Scan Mass Spectrometry.

Niklas Kleinenkuhnen,Silke C Gerlich,Stanislav Kopriva,Felix Büchel,Sabine Metzger

doi:10.3389/fpls.2019.00885

Niklas Kleinenkuhnen, Silke C Gerlich + Show 3 more

Open Access

https://doi.org/10.3389/fpls.2019.00885

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Abstract

Sulfur is present in plants in a large range of essential primary metabolites, as well as in numerous natural products. Many of these secondary metabolites contain sulfur in the oxidized form of organic sulfate. However, except of glucosinolates, very little is known about other classes of such sulfated metabolites, mainly because of lack of specific and quantitative analytical methods. We developed an LC-MS method to analyze sulfated flavonoids, a group of sulfated secondary metabolites prominent, e.g., in plants of the genus Flaveria. The method uses a linear gradient of methanol/formic acid in water on a Restek Raptor C18 Core-Shell column for separation of the compounds. The sulfated flavonoids are detected by mass spectrometry (MS) in a negative mode, using a neutral loss of 80 Da after a collision induced dissociation. With this method we were also able to quantify the sulfated flavonoids. We could detect all (mono)sulfated flavonoids described before in Flaveria plus a number of new ones, such as isorhamnetin-sulfate-glycoside. In addition, we showed that sulfated flavonoids represent a substantial sulfur pool in Flaveria, larger than the thiols glutathione and cysteine. The individual species possess different sulfated flavonoids, but there is no correlation between the qualitative pattern and type of photosynthesis. Similar to other sulfur-containing secondary compounds, the concentration of sulfated flavonoids in leaves is reduced by sulfur starvation. The new LC-MS method will enable qualitative and quantitative detection of these secondary metabolites in plants as a pre-requisite to addressing their functions.

Highlights

Sulfur (S) is an essential nutrient for all life forms
Prior to optimizing the instrument parameters, it was necessary to determine whether the loss of the sulfate group of sulfated flavonoids upon collision-induced dissociation (CID) is the main fragmentation channel
With the neutral loss scan based method, we identified these patterns in a single experiment but we achieved a relative quantification of the detected sulfated flavonoids together with an absolute quantification of quercetin-3-sulfate within this single experiment

Summary

Introduction

Sulfur (S) is an essential nutrient for all life forms In plants it is present in a plethora of metabolites of primary and secondary metabolism, most prominently in the amino acids cysteine and methionine. In the majority of these metabolites S is present in a reduced form, i.e., as a thiol, some compounds, mainly various secondary metabolites, contain S in an oxidized form, i.e., as a sulfate (Takahashi et al, 2011). For both groups of metabolites, the source of S. The control of S partitioning in the primary (reduced) or secondary (oxidized) metabolism is achieved by the interplay of the corresponding enzymes, APS reductase and APS kinase (Mugford et al, 2011)

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