Comprehensive multidimensional separations of peptides using nano-liquid chromatography coupled with micro free flow electrophoresis.

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (µFFE) separation. Since µFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in µFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC × µFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC × µFFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC × µFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC × LC.

Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies.

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94% of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ∼2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.

Using buffer additives to improve analyte stream stability in micro free flow electrophoresis.

Micro free flow electrophoresis (microFFE) is a separation technique that continuously separates analyte streams as they travel through an electric field applied perpendicularly to the flow in a microdevice. Application of the technique has been limited by the generation of electrolysis bubbles at the electrodes, which results in unstable flow paths through the device. The current paper introduces the use of surfactants and nonaqueous solvents in the carrier buffer as a means of increasing stability of separated analyte streams. Adding surfactant or nonaqueous solvents lowers the surface tension of the carrier buffer, which we hypothesize promotes the formation of smaller electrolysis bubbles. A 6-fold improvement in the standard deviation of analyte stream position was observed upon addition of 10 mM SDS. Likewise, an approximately 12-fold improvement in stability was observed upon addition of 300 microM Triton X-100. Similar stability improvements were found in carrier buffers containing nonaqueous solvents. An 8-fold improvement in stability was found with a carrier buffer containing 50% methanol and a 6-fold improvement was found with a carrier buffer containing 37.5% acetonitrile. Long term use was demonstrated with a carrier buffer containing 300 microM Triton X-100 in which separated analyte streams remained stable for nearly two hours.