Microfluidic technologies hold high promise and emerge as a potential molecular tool to facilitate the progress of fundamental and applied biomedical researches by enabling miniaturization and upgrading current biological research tools. In this review, we summarize the state of the art of existing microfluidic technologies and its’ application for characterizing bio- physical properties of individual cells. Microfluidic devices offer significant advantages and ability to handle in integrating sam- ple processes, minimizing sample and reagent volumes, and increased analysis speed. Therefore, we first present the basic concepts and summarize several achievements in new coupling between microfluidic devices and mass spectrometers. Secondly, we discuss the recent applications of microfluidic chips in various biological research field including cellular and molecular level. Finally, we present the current challenge of microfluidic technologies and future perspective in this study field.
Studies on the interactions of amyloidogenic proteins with trace metals, such as copper, have indicated that the metal ions perform a critical function in the early oligomerization process. Herein, we investigate the effects of Cu(II) ions on the active sequence regions of amyloidogenic proteins using electrospray ionization mass spectrometry (ESI-MS) and collision induced dissociation tandem MS (CID-MS/MS). We chose three amyloidogenic peptides NNQQNY, LYQLEN, and VQIVYK from yeast prion like protein Sup35, insulin chain A, and tau protein, respectively. [Cu-peptide] complexes for all three peptides were observed in the mass spectra. The mass spectra also show that increasing Cu(II) concentrations decrease the population of existing peptide oligomers. The tandem mass spectrum of NNQQNY shows preferential binding for the N-terminal region. All three peptides are likely to appear to be in a Cu-monomer-monomer (Cu-M-M) structure instead of a monomer-Cu-monomer (M-Cu-M) structure.
DC23, a triazolothione resorcinol analogue, is known to inhibit heat shock protein 90 and pyruvate dehydrogenase kinase which are up-regulated in cancer and diabetes, respectively. This study was performed to elucidate the metabolism of DC23 in human liver microsomes (HLMs). HLMs incubated with DC23 in the presence of uridine 5’-diphosphoglucuronic acid (UDPGA) and/or β-nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the formation of four metabolites, M1- M4. M1 was identified as DC23-N-Oxide, on the basis of LC-MS/MS analysis. DC23 was further metabolized to its glucuronide conjugates (M2, M3, and M4). In vitro metabolic stability studies conducted with DC23 in HLMs revealed significant glucuron- ide conjugation with a t 1/2 value of 1.3 min. The inhibitory potency of DC23 on five human cytochrome P450s was also investi- gated in HLMs. In these experiments, DC23 inhibited CYP2C9-mediated tolbutamide hydroxylase activity with an IC 50 value of 8.7 µM, which could have implications for drug interactions.
A quantitation method for free amino acids in human serum was developed using a stepwise-dilution method and a bimodal cation exchange (CEX)/hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry system equipped with an electrospray ionization source (ESI/MS/MS). This method, which was validated using quality control samples, was optimized for enhanced selectivity and sensitivity. Dithiothreitol (DTT) was used as a reducing agent to prevent the oxida- tion of a serum sample (50 µL), which was then subjected to stepwise dilution using 3, 30, and 90 volumes of acetonitrile con- taining 0.1% formic acid. Chromatographic separation was performed on an Imtakt Intrada Amino Acid column (50 mm × 3 mm, 3 µm) in mixed mode packed with CEX and HILIC ligands embedded in the stationary phase. Underivatized free amino acids were eluted and separated within 10 min. As a result of the validation, the precision and accuracy for the inter- and intraday assays were determined as 2.11-11.51% and 92.82-109.40%, respectively. The lowest limit of quantification (LLOQ) was 0.5- 4.0 µg/mL and the matrix effect was 80.22-115.93%. The proposed method was successfully applied to the quantitative analysis of free amino acids in human serum.
Farinomalein is a maleimide-bearing compound well known for its anti-fungal activity. In the present study, synthesis of farinomalein is achieved via Stobbe condensation followed by Haval-Argade contrathermodynamic rearrangement. Kinetically driven Stobbe condensation followed by condensation with beta-alanine reveals formation of two isomers of farinomalein. This article describes application of LC-MS/MS in structure elucidation of farinomalein 1 and its isomers 2 and 3 encountered in its synthesis. The proposed distinct fragmentation pathway is supported by rational organic reaction mechanism. These fragmentation pathways are significant for analytical method development of farinomalein in near future. The structures of farinomalein 1 and its isomers 2 and 3 have been assigned undisputedly.