Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.

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Rose tea is a kind of popular beverage in many Asian countries. In this study, the contents of volatiles, polysaccharides and total polyphenols in three species of Chinese rose tea infusions [Kuishui Rose (KSR), Pinying Rose (PYR) and Jinbian Rose (JBR)] were determined, respectively. Meantime, the volatiles from those three tea infusions were analyzed by GC‐MS, and their antioxidant activities were tested by 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging assay. As results, 17, 15, and 20 components were identified in their volatiles, respectively. The contents of volatiles, polysaccharides and total polyphenols, and the antioxidant activity of PYR tea infusion were higher than those of other two tea infusions. To elucidate the contributions of those main constituents in rose tea for its activity, correlation coefficients between them were analyzed, displaying that the polysaccharides and polyphenols are the main antioxidants in rose tea infusion, whose correlation coefficients were greater than 0.9. Practical applications As a kind of beverage with health care function, the activities and active ingredients of rose tea have mainly focused on polyphenols and their antioxidant so far. The current studies on the contents of volatiles, polysaccharides and total polyphenols and the antioxidant activities of rose tea infusions from three species in China could provide an useful comparison of the difference of antioxidant activities and constituents in those three rose teas and a basic data for quality control of rose tea.