A mass spectrometric method for quantifying C3 and C6 phosphorylation of starch

Area of Science:

• Biochemistry
• Analytical Chemistry
• Plant Science

Background:

• Starch phosphorylation occurs at C3 and C6 positions, catalyzed by distinct dikinase enzymes.
• Quantifying C6 phosphorylation is straightforward, but C3 phosphorylation measurement is challenging.
• Accurate assessment of starch phosphorylation is crucial for understanding its properties and function.

Purpose of the Study:

• To develop a mass spectrometric (MS) method for simultaneous quantification of glucose 3-phosphate and glucose 6-phosphate from hydrolyzed starch.
• To establish an efficient method for determining starch phosphorylation levels in various potato lines.
• To investigate the impact of cold storage on starch phosphorylation in potato tubers.

Main Methods:

• Development of a liquid chromatography-mass spectrometry (LC/MS) method using normal-phase hydrophilic interaction liquid chromatography.
• Quantification by selective reaction monitoring (SRM) with external standards.
• Application of the MS method to analyze starch phosphorylation in diverse potato genotypes and assess stability during cold storage.

Main Results:

• The developed LC/MS method successfully distinguished and quantified both glucose 3-phosphate and glucose 6-phosphate in a single run.
• Significant threefold differences in starch phosphorylation levels were observed between high- and low-phosphate potato lines.
• Cold storage of potato tubers for up to 24 weeks showed minimal impact on starch phosphorylation levels.

Conclusions:

• Mass spectrometry provides an effective and efficient approach for determining both C3 and C6 starch phosphorylation.
• The MS method enables detailed analysis of starch phosphorylation variations in different potato varieties.
• Starch phosphorylation in potato tubers is relatively stable under prolonged cold storage conditions.