Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry

AbstractConsecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, β-casein and α-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation

Characterization and Classification of Pseudo-Stationary Phases in Micellar Electrokinetic Chromatography Using Chemometric Methods

Two types of chemometric methods, principal component analysis (PCA) and cluster analysis, are employed to characterize and classify a total of 70 pseudostationary phases (54 distinct systems and 16 decoy systems) in micellar electrokinetic chromatography (MEKC). PCA excels at removing redundant information for micellar phase characterization and retaining principal determinants for phase classification. While PCA is useful in the characterization of micelle selectivities, it is ineffective in defining the grouping of micellar phases. Hierarchical clustering yields a complete dendrogram of cluster structures but provides only limited cluster characterizations. The combination of these two chemometric methods leads to a comprehensive interpretation of the micellar phase classification. Moreover, the k-means analysis can further discern subtle differences among those closely located micellar phases. All three chemometric methods result in similar classifications with respect to the similarities and differences of the 70 micelle systems investigated. These systems are categorized into 3 major clusters: fluoro-surfactants represent cluster I, identified as strong hydrogen bond donors and dipolar but weak hydrogen bond acceptors. Cluster II includes sulfonated acrylamide/acrylate copolymers and surfactants with trimethylammonium head groups, characterized by strong hydrophobicity (<i>v</i>) and weak hydrogen bond acidity (<i>b</i>). The last cluster consists of two subclusters: clusters III and IV. Cluster III includes siloxane-based polymeric micelles, exhibiting weak hydrophobicity and medium hydrogen bond acidity and basicity (<i>a</i>), and the cluster IV micellar systems are characterized by their strong hydrophobicity and medium hydrogen bond acidity and basicity but rather weak dipolarity. Cluster III differs from cluster IV by its slightly weaker hydrophobicity and hydrogen bond donating capability. The classification by chemometric methods is in good agreement with the classification by the micellar selectivity triangle (MST) (Fu, C.; Khaledi, M. G. J. Chromatogr., A 2009, 1216, 1891−1900)

Perfluoro-Alcohol-Induced Complex Coacervates of Polyelectrolyte–Surfactant Mixtures: Phase Behavior and Analysis

Perfluorinated alcohols and acids such as hexafluoroisopropanol (HFIP), trifluoroethanol, trifluoroacetic acid, pentafluoropropionic acid, and heptafluorobutyric acid induce coacervation and phase separation in aqueous solutions of a wide variety of individual and mixed amphiphiles [Khaledi Langmuir 2013, 29, 2458]. This paper focuses on HFIP-induced complex coacervate formation in the mixtures of anionic polyelectrolytes, such as sodium salt of poly­(methacrylic acid) (PMA) or poly­(acrylic acid) (PAA) and cationic surfactants of alkyltrimethylammonium bromides. In purely aqueous media and over a wide concentration range, mixtures of PMA and CTAB form the catanionic complex (CTA⁺PM^–) that is insoluble in water (white precipitate). Upon addition of a small percentage of HFIP, the mixture goes through phase transition and formation of two distinctly clear liquid phases. The phase diagram for the HFIP–PMA–CTAB coacervate system was studied. The coacervate volume was determined as a function of system variables such as charge ratio as well as total and individual concentrations of the system components. These results, combined with the chemical composition analysis of the separated aqueous top-phase and coacervate bottom-phase, shed light on the coacervation mechanism. The results suggest that exchange of counterions and ion-pair formation play critical roles in the coacervation process. This process facilitated by HFIP through solvation of the head groups and dehydration of the hydrophobic moieties of the catanionic complex. Because of the presence of HFIP, coacervation occurs over a wide range of concentrations and charge ratios of the oppositely charged polyelectrolyte and surfactant