We developed a capillary LC/MS/MS-based approach to monitor intracellular kinase activities. Selected reaction monitoring (SRM) mode was employed to quantitate a kinase substrate-digested peptide phosphorylated by kinase or kinase-digested peptides containing phosphosites which regulate the kinase activities. Ten kinases in EGFR-MAPK signaling pathway were targeted for the SRM assay and the experimental conditions such as the selection of target phosphopeptides, SRM transitions, LC parameters and sample pre-treatment steps were optimized. The validation study on accuracy, precision, linearity, limit of detection and limit of quantitation was carried out to confirm the capability for measuring the kinase activities through the phosphopeptide quantities in the biological samples. Finally, we applied this SRM assay to kinase activation dynamics induced by pervanadate, a tyrosine phosphatase inhibitor, in HeLa cells. As a result, it was found that 6 kinases out of 10 were activated, which were consistent with those by conventional Western blotting using phosphosite-specific antibodies. Since this SRM assay can be extended to kinome-wide analysis, it will be useful to unveil the entire signaling network in cells.
We simultaneously quantified fecal bile acids (BAs) and short-chain fatty acids (SCFAs) florescence labeled with 9-chloromethylanthracene using high-performance liquid chromatography-fluorescence (HPLC-FL) and developed an inexpensive and highly accurate method for determining the ratio of BAs and SCFAs in colorectal cancer patients and healthy controls. Samples were extracted with hexane/ether, and extractants were measured using HPLC-FL. The healthy subject group included 17 men and 21 women, whereas the colorectal cancer group included patients with cancer in the rectum (3 men, 2 women), sigmoid colon (3 men, 2 women), and ascending colon (2 men, 3 women). The contribution rate of determination for calibration curves was >0.99, and the additional recovery rate was 67.2%–107% for the simultaneous quantification of fecal BAs and SCFAs using HPLC-FL. Intra-day and inter-day variations in the control feces ranged 2.4%–5.1% and 3.1%–9.2%, respectively. The proportion of primary BAs to total BAs was higher in the colorectal cancer group (87.3%) than in the healthy subject group (67.9%). A significant difference was observed in the ratio of BAs to butyric acid between healthy subject and colorectal cancer groups. BA levels were higher in the colorectal cancer group. Thus, the ratio of total BAs to butyric acid may be a better predictor of colon cancer than BAs or SCFAs alone.
Continuous counter-current foam separation (CCFS) is a method for the recovery of valuable metals with high selectivity. It was developed as an organic solvent-free method, and the interaction of surfactants and metal ions in aqueous solution is a key precondition for the successful recovery. In this study, the interactions between the anionic complex of palladium(II) chloride and N,N’-dimethyl-N,N’-di-n-hexyl-thiodiglycolamide (MHTDA), which is an extraction agent for palladium(II), were investigated by capillary electrophoresis, in the presence of surfactants (sodium dodecyl sulfate (SDS) or polyoxyethylene mono-4-octylphenyl ether (POOPE)). The addition of MHTDA to an electrophoretic media containing SDS, resulted in electropherograms composed of two or three peaks. On the other hand, in the case of an electrophoretic media containing POOPE, the elimination of palladium (II) peak was observed due to MHTDA addition. This behavior suggested that the palladium-MHTDA neutral complex is rapidly captured in the POOPE micelles compared with the SDS micelles, and the use of POOPE is effective for the successful recovery of palladium(II) in CCFS.
Quantitative analysis of monosaccharides or glycoprotein glycans by high-performance liquid chromatography (HPLC) often involves labeling of the saccharide aldehyde groups with fluorescent tags to enhance sensitivity and selectivity. However, the methods required to remove the large excess of labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods often hinder the quantitative analysis of the labeled samples. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminobenzoic acid (2-AA)-labeled monosaccharides or oligosaccharides using a ten-port valve and mini columns. An online purification system using a combination of short HLB columns with the valve was proposed for the analysis of 2-AA-labeled monosaccharides utilizing reversed-phase modes. In the analysis of 2-AA-labeled glycans derived from glycoproteins, a short CN column with the valve was proposed utilizing hydrophilic interaction liquid chromatography (HILIC) modes. Optimized conditions enabled the direct injection of the diluted labeling reaction mixture into the chromatographic system without any prior removal of the excess labeling reagents. These methods were successfully applied to the analysis of various monosaccharides and N-linked glycans released from specific glycoproteins.
Therapeutic drug monitoring (TDM) is a clinical practice that designs personalized medication for patients with blood concentrations of the drug. TDM approach is used for many drugs, including immunosuppressant, antifungal, antiarrhythmic, and anti-cancer drugs. Combination therapies are often adopted in TDM. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a useful analytical method in such cases. However, the development of a simultaneous LC-MS/MS analytical method is difficult owing to the differences in MS sensitivity and the therapeutic range of each drug. In order to avoid saturation of the detector, in-source collision induced dissociation (CID) was used to reduce the ion inlet. In this study, we investigated the in-source CID behavior of 13 compounds of drugs and metabolites in TDM practice. As a result, all compounds provided a sharp reduction of ion inlet over the threshold ion guide voltage. In addition, a shift to the higher concentration of the calibration range was observed according to such changes. The intensity and linearity data in this study that all 13 drugs could be analyzed under in-source CID conditions simultaneously. These results might be useful for TDM of combination therapy in clinical practice.
To achieve highly sensitive analyses of cationic analytes with simple experimental procedures in non-aqueous capillary electrophoresis (NACE), large-volume sample stacking with an electroosmotic flow pump (LVSEP) was performed in dynamically polymer coated capillaries. As the dynamic coating polymers, cationic polybrene (PB), and neutral polymer, (hydroxypropyl)methyl cellulose (HPMC), were employed in methanolic media to reverse and suppress the electroosmotic flow. In the analysis of cationic amines, good enrichments were attained with the sensitive enhancement factor (SEF) of 70 and 687 for benzylamine and 1-(1-naphthyl)ethylamine, respectively, with a methanolic running solution containing 1.0% HPMC and 0.5% PB. In the NACE-LVSEP analysis of Ru(bpy)32+ and Ru(phen)32+, furthermore, the values of SEF were almost 40 with almost no-loss of the resolution.
Chiral amino acids in fermented products including Japanese traditional black vinegar, a processed cheese and nam pla were determined using an on-line two-dimensional (2D) HPLC-MS/MS system. As the target amino acids, Ala, Asp, Glu, Leu, Pro and Ser were selected. Prior to the HPLC separation, the fermented products were appropriately homogenized and deproteinized, then amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Using the 2D-HPLC system, the target NBD-amino acids were individually purified by a microbore reversed-phase column in the first dimension and further separated by a narrowbore enantioselective column in the second dimension. The detection was performed by a fluorescence detector and also by a tandem mass spectrometer. Compared to the 2D-HPLC with fluorescence detection, the target chiral amino acids in complicated real world matrices were successfully determined using the 2D HPLC-MS/MS system without interference by the co-elution of unknown intrinsic compounds. In all the tested fermented products, various D-amino acids were observed, and the obtained values were 0.02-6.21 mmol/L (%D=0.7-29.2%) in the black vinegar, 0.02-0.73 µmol/g (0.2-24.8%) in the processed cheese and 0.07-2.30 mmol/L (0.5-23.5%) in the nam pla.