We propose a novel single-cell chemical proteomics (SCCP) strategy to profile low-abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity-based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser-induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation.

Chiral recognition of dansyl enantiomeric amino acids by microfluidic open tubular CEC (μOTCEC) with fluorescence detection was demonstrated. Avidin was employed as the chiral selector immobilized on the microchannel wall, which functioned as the chiral stationary phase (CSP) by physical adsorption. The condition of CSP on the glass wall was characterized using field emission SEM. Results indicated that avidin was homogenously distributed on the microchannel surface. Two parameters that played essential roles in μOTCEC for chiral recognition were investigated. Buffer pH could greatly change the amount of adsorption of avidin on the channel wall by altering the electrostatic attraction between them. Methanol, the organic additive to the running buffer, was also found significant for controlling the quality of the μOTCEC chiral separation by regulating the hydrophobic interaction between the enantiomers and the CSP. Under the optimized conditions, four dansyl racemic amino acids were then successfully separated by μOTCEC within 100 s with resolutions of 2.43, 1.88, 3.01 and 2.65 for dansyl-Ser, dansyl-Met, dansyl-Thr and dansyl-Val, respectively. Furthermore, a comparison with microfluidic CZE was investigated demonstrating that μOTCEC was a promising method for rapid chiral recognition.

Membrane proteins are generally of natural low abundance and insoluble to aqueous solutions. Thus, investigation of membrane proteins is relatively hampered since efficient separation of membrane proteins are rather challenging. Microseparation approaches certainly play an essential role in fulfilling such demanding investigations due to their significant advantages of high throughput, reduced separation time, and low sample consumption. In this review, recent developments of microseparation are documented with aspects of three key separation methods, including CE, micro-LC and microchip. Preparation of membrane proteins prior to separation is also discussed, including solubilization and preconcentration. Certainly, more applications of microseparation approaches in membrane protein separations can be expected in the near future.

Biogenic amines are a group of biological molecules derived from the enzymatic decarboxylation of natural amino acids. They can be found in a variety of foods and some of them are involved in essential cellular pathways regulating cellular functions. To address the issues raised by conventional detection methods for biogenic amines, such as laborious sample preparation and limited sensitivity, a new micellar electrokinetic chromatography scheme was developed based on multiphoton excitation fluorescence (MPEF) detection. Six FITC-labeled biogenic amine species were used for the evaluation of this MEKC–MPEF method in comparison to single photon excitation fluorescence detection. The results indicated that MEKC–MPEF had superior resolution with a detection volume as low as aL. Quantitative analysis of varying concentrations of biogenic amine species has also been achieved suggesting a ymole mass detection limit, a linear dynamic range of about two orders of magnitude, and 95–105% recoveries. Furthermore, the biogenic amine profile of decayed oriental crucian carps was successfully determined and quantified using this new method.