The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in a2-3 and a2-6 linkages, while these glycans remain attached to peptides.
To examine whether ferulic acid (FA) could protect plants from dehydration stress and to investigate a mechanism for the protection, cucumber seedlings were pretreated with 0.5 mM FA for 2 d and then were exposed to dehydration induced by 10 % polyethylene glycol 6000. After pretreatment with FA, the activities of antioxidant enzymes (catalase, superoxide dismutase, and quaiacol peroxidase) in leaves were higher than under dehydration treatment alone which was in accordance with the increased transcript levels of respective genes. Moreover, the combination of FA pretreatment and dehydration reduced the content of superoxide radical, hydrogen peroxide, and malondialdehyde, and increased the relative water content and content of FA, proline, and soluble sugars in comparison with dehydration alone. We propose that pretreatment with FA protects cucumbers against dehydration stress by decrease of lipid peroxidation due to activation of antioxidant enzymes and by increase of proline and soluble sugar content in leaves.
To investigate the physiological mechanisms of drought stress mitigated by exogenous cinnamic acid (CA), cucumber seedlings were pretreated with 50 lM CA for 2 days and then were subjected to 10% polyethylene glycol (PEG) 6000. We examined if CA protects plants from PEGinduced drought stress, and whether the protective effect is related to antioxidant and lipid-peroxidation regulation. 2 days of CA application enhanced the activities of guaiacol peroxidase (GPX), glutathione peroxidase (GSH-Px), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and increased the levels of ascorbate, proline, soluble sugar, vanillic acid (VA) and CA in leaves. After CA-pretreated leaves were exposed to drought, the activities of superoxide dismutase (SOD), catalase, ascorbate peroxidase, monodehydroascorbate reductase, GPX, GSH-Px, DHAR and GR were higher than under drought stress alone, while the levels of reduced glutathione, ascorbate, proline, soluble sugar, VA and CA in leaves were more. The combination of exogenous CA and drought led to higher transcript levels of Cu/Zn-SOD and Mn-SOD genes and decreased contents of malonaldehyde and hydrogen peroxide, but drought had adverse effects on them. Furthermore, the combined effects of exogenous CA and drought made 61.67% leaf edges dried, while drought resulted in 95.83% withered leaves. We conclude CA pretreatment leads to higher contents of CA and VA in drought-stressed leaves and thereby results in higher antioxidant activities directly or indirectly via proline and soluble sugar, thus increasing drought tolerance of cucumber. We also propose soluble sugar can reduce reactive oxygen species and decrease lipid peroxidation when exogenous CA mitigates drought stress.
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