Rapid and sensitive quantification of C3- and C6-phosphoesters in starch by fluorescence-assisted capillary electrophoresis

TL;DR

This paper presents a rapid, sensitive capillary electrophoresis method for quantifying C3- and C6-phosphoesters in starch, enabling high-throughput analysis of starch phosphorylation with fg sensitivity.

Contribution

A novel capillary electrophoresis technique for simultaneous, high-sensitivity measurement of Glc-3-P and Glc-6-P in starch, improving quantification efficiency.

Findings

Glc-3-P content varies independently of Glc-6-P in potato lines.

The method detects phosphoesters at the fg level, suitable for microgram samples.

Alterations in starch phosphorylation relate to genetic modifications.

Abstract

Phosphate groups are naturally present in starch at C3- or C6-position of the glucose residues and impact the structure of starch granules. Their precise quantification is necessary for understanding starch physicochemical properties and metabolism. Nevertheless, reliable quantification of Glc-3-P remains laborious and time consuming. Here we describe a capillary electrophoresis method for simultaneous measurement of both Glc-6-P and Glc-3-P after acid hydrolysis of starch. The sensitivity threshold was estimated at the fg scale, which is compatible with the analysis of less than a $\mu$g of sample. The method was validated by analyzing antisense potato lines deficient in SBEs, GWD or GBSS. We show that Glc-3-P content is altered in the latter and that these variations do not correlate with modifications in Glc-6-P content. We anticipate the method reported here to be an efficient tool for high throughput study of starch phosphorylation at both C3- and C6-position.