A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

Evaluation of solid-phase extraction sorbents for purification of oligosaccharides and glycans derivatized by positively charged labels followed by capillary electrophoretic analysis.

The sample preparation including labeling and clean-up represents a key analytical step in the analysis of oligosaccharides and glycans by either chromatographic or electrophoretic separation methods. Although the majority of labeling has been performed by neutral and/or negatively charged tags, the introduction of a positive charge into the saccharide molecule can significantly improve the analysis, especially with mass spectrometry detection. In this work, we present the evaluation of five solid-phase extraction sorbents differing in extraction chemistry for the clean-up and concentration of positively labeled maltooligosaccharides from the reaction mixtures. Maltooligosaccharides containing four to seven glucose units were labeled by cationic tags (2-aminoethyl)trimethylammonium chloride and (carboxymethyl)trimethylammonium chloride hydrazide and the extraction conditions were optimized followed by electrophoretic analysis with conductivity detection. The effects of the solid-phase extraction sorbent chemistry, extraction conditions, and sample composition are discussed. All tested sorbents were capable of cleaning up maltooligosaccharides from the reaction mixtures to some extent after optimization of the solid-phase extraction procedure (51.9%-98.9% recovery). The best-rated amide-based sorbent was used to process the sample of N-linked glycans enzymatically released from ribonuclease B.

Synthesis and application of BODIPY-based fluorescent labeling tag for oligosaccharide and N-linked glycan analysis by high-performance liquid chromatography with fluorescence detection.

BACKGROUND: Glycosylation analysis is still challenging, not only because of the extreme structure complexity and conjugation diversity of glycans but also because of instrumental aspects such as the sensitivity limits of analyses. Therefore, glycan analysis by chromatographic methods is very often combined with fluorescence detection in addition to MS. The majority of fluorescent labeling employed before LC separation is based on 2-aminobenzamide, which has several disadvantages such as low labeling yield, poor fluorescence properties, and MS ionization efficiency. Therefore, even after several decades of development of new labels, there is still a need for new labeling tags with improved characteristics. RESULTS: We present the application of a newly synthesized fluorescent label designed for oligosaccharide and glycan analysis by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). The novel hydrazide derivative of dipyrrometheneboron difluoride (BODIPY) was synthesized from 2,4-dimethylpyrrole, methyl succinyl chloride, and boron trifluoride etherate followed by a reaction with hydrazine. The synthesized label was characterized by several analytical methods including NMR, UV/Vis and fluorescence spectroscopy, and mass spectrometry. The labeling reaction via hydrazone formation chemistry was optimized by labeling of maltooligosaccharide standards. The analysis of maltohexaose labeled by BODIPY-hydrazide followed by HPLC/FLD analysis provided the limit of detection in the low tens of femtomole. The presented method based on fluorescence detection is at least 30 times more sensitive than the standard approach employing labeling by 2-aminobenzamide. In addition, the labeling method by BODIPY-hydrazide was used for N-linked glycan profiling of several glycoproteins (ribonuclease B, immunoglobulin G) by RP-HPLC/FLD as well as HILIC/FLD analysis. SIGNIFICANCE: This work represents the design, synthesis, and application of a new fluorescent label based on the BODIPY core and hydrazone formation chemistry for oligosaccharide and glycan analysis by HPLC/FLD. The proposed approach significantly improved the oligosaccharide and glycan analysis in comparison to the commonly used procedure employing 2-aminobenzamide.