Quantification of Low-Abundant Phosphorylated Carbohydrates Using HILIC-QqQ-MS/MS

Abstract

Phosphorylated carbohydrates are central metabolites involved in key plant metabolic pathways, such as glycolysis and central carbon metabolism. Such pathways influence plant growth, development, and stress responses to environmental changes, and ultimately, reflect the plant's energy status. The high polarity of these metabolites, the variety of isomeric structures (e.g., glucose-1-phosphate (G1P)/fructose-6-phosphate (F6P)/mannose-6-phosphate (M6P)/G6P, sucrose-6-phosphate (S6P)/T6P), and rapid metabolic turnover makes their analysis particularly challenging. In this chapter, we describe the use of a set of known phosphorylated carbohydrates to develop and validate a hydrophilic interaction chromatography (HILIC) triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of G1P, F6P, M6P, G6P, S6P, T6P, and the sugar nucleotide uridine 5-diphospho-glucose (UDPG). We present detailed information regarding HILIC column chemistry and practical considerations when coupling it with a QqQ-MS system.

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…However, due to the microheterogeneity and complexity of the glycan structures, isomeric separation of glycans is still lacking. Several LC-MS techniques have been employed to facilitate the isomeric separation of glycans including RP [80,81], HILIC [82,83], porous graphitic carbon (PGC) [84,85], and mesoporous graphitic carbon (MGC) [86]. This section discusses the most recent LC and CE methods that assist in the isomeric characterization and separation of glycans.…”