Academic Journal
Starch phosphorylation—A needle in a haystack
| Title: | Starch phosphorylation—A needle in a haystack |
|---|---|
| Authors: | Julia Compart, Ardha Apriyanto, Joerg Fettke |
| Source: | Plant Methods, Vol 20, Iss 1, Pp 1-11 (2024) |
| Publisher Information: | BMC, 2024. |
| Publication Year: | 2024 |
| Collection: | LCC:Plant culture LCC:Biology (General) |
| Subject Terms: | Starch phosphorylation, Starch, Phosphoglucans, Glucan, α-glucan, water dikinase, Mass spectrometry, Plant culture, SB1-1110, Biology (General), QH301-705.5 |
| More Details: | Abstract Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities. |
| Document Type: | article |
| File Description: | electronic resource |
| Language: | English |
| ISSN: | 1746-4811 |
| Relation: | https://doaj.org/toc/1746-4811 |
| DOI: | 10.1186/s13007-024-01237-9 |
| Access URL: | https://doaj.org/article/5b542da192944aef9161187e2fbb3b9d |
| Accession Number: | edsdoj.5b542da192944aef9161187e2fbb3b9d |
| Database: | Directory of Open Access Journals |
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| ISSN: | 17464811 |
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| DOI: | 10.1186/s13007-024-01237-9 |
| Published in: | Plant Methods |
| Language: | English |