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Fluorescence-based analysis of the contents of a single cell may rely upon fluorophore derivatization or the native fluorescence of the analyte itself. The former strategy—called laser-induced native fluorescence, or LINF—can be productively combined with capillary electrophoresis for cellular analysis. A robust system for CE-LINF has been previously described by Sweedler and colleagues, which consists, in part, of a spectrograph for wavelength resolution and a charge-coupled device (CCD) for detection. The ability to obtain wavelength-resolved spectra allows species with overlapping migration times to be distinguished. This power comes at substantial financial expense; therefore Sweedler and colleagues have returned to the problem of CE-LINF system design and now describe a more economical approach. In this new CE-LINF setup, photomultiplier tubes (PMT) are used for detection and beam-splitters set the stage for multi-channel monitoring of fluorescence. The system is integrated in such a way as to synchronize the HeAg laser pulses with fluorescence detection, which benefits sensitivity by minimizing capture of stray signals. The authors describe the application of this newly designed apparatus to the detection of neurotransmitters such as serotonin and dopamine. To validate their instrumentation, Lapainis et al., turned to Aplysia and the pond snail Lymnaea stagnalis, which both have neuronal cells that are well characterized. In the former, a serotonergic neuron and a dopaminergic counterpart were dissected and the single cell was suspended in a citrate buffer and then injected into the capillary. Comparison of the channel-separated electropherograms confirmed the presence of serotonin and dopamine, respectively. Although the migration time of dopamine in a CNS subcompartment of Aplysia differed from the signal obtained with the freshwater mollusk, the identity of the neurotransmitter could be confirmed on the basis of the characteristics of traces in the relevant channels. These results confirm that this new CE-LINF approach is applicable to the detection of neurotransmitters in the low nanomolar range, and should prove useful for studying intercellular signaling of neuronal circuits.
- Lapainis et al. 2006. A multi-channel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons. Analytical and Bioanalytical Chemistry [Epub ahead of print, September 20, 2006].
A Perfect FitA researcher who has identified a promising antibody, but who needs to discern the exact location of the epitope in the target antigen, faces a potentially time-consuming process. It is easy to see why the classical wet-lab approach of deriving a crystal structure of the antibody bound to its antigen would be unappealing. Fortunately, phage display has proven to be a powerful (and more convenient) approach. In this strategy, peptides that bind with high affinity to the antibody are analyzed and the consensus sequence is found. More often than not, the sequence is a mimotope, a sequence which mimics the true epitope's charge and shape rather than its exact sequence. Clearly, computational methods would be extremely useful in order to match the mimotope with the native epitope; however, no published software has hit the sweet spot of offering flexible features while remaining platform independent and freely available. Now, Huang et al., introduce MIMOX, a webbased tool for epitope mapping from phage display data. MIMOX begins with the mimotope sequences that have been determined via the phage display process; these are aligned using ClustalW and are processed in order to derive a consensus sequence. The software then combines this piece of information with a PDB file containing the antigen molecule's structure and provides the user with promising epitope candidates. Users can easily visualize mapping results in the context of the antigen's overall structure, and may peruse data regarding the surface accessibility of each candidate. MIMOX is implemented in Perl, is freely available for use by academic and commercial researchers alike, and can be accessed online at web.kuicr.kyoto-u.ac.jp/∼hjian/mimox.
- Huang et al. 2006. MIMOX: a web tool for phage display based epitope mapping. BMC Bioinformatics 7:451.
The Inside StorySubproteomes have been a trendy area of investigation, partly due to genuine curiosity about the protein repertoire of a given cellular component, and also because the task of defining all of the proteins in a whole cell is beyond the reach of current technology. A recent study examining the subproteome of the inner mitochondrial membrane offers both interesting biological findings and notable methodological information. McDonald et al., analyzed how the choice of one of three popular techniques for protein separation affected the character and number of unique protein identifications obtained. The three methods studied were two-dimensional gel electrophoresis (2-DGE), one-dimensional reversed-phase HPLC (1-DLC), and two-dimensional liquid chromatography (2-DLC). In each case, protein identifications were done by mass spectrometry (MALDI-TOF for the gel-based method and ESI MS/MS for the liquid chromatography techniques). The number of nonredundant proteins identified ranged from 77 (2-DGE) to 146 (2-DLC) to 230 (1-DLC). The coincidence between all three methods was just 7%, so the total number of unique proteins found was 347. The 2-DGE approach stumbled in the identification of particularly hydrophobic proteins, or those with extreme pI values. Both liquid chromatography approaches were especially adept at finding proteins of low molecular weight (<30 kDa) and basic pI values. Compared with previously published descriptions of the total mitochondrial membrane proteome or an inner mitochondrial membrane subproteome, this new study from McDonald et al., includes 134 additional proteins, most of which were found only with the use of 1-DLC. Careful examination of these findings should be of interest to researchers looking to select a platform or platforms for comprehensive subproteome analysis.
- McDonald et al. 2006. Expanding the subproteome of the inner mitochondria using protein separation technologies: one and two-dimensional liquid chromatography and two-dimensional gel electrophoresis. Molecular & Cellular Proteomics [Epub ahead of print, October 20, 2006].
How Sweet It IsLectin-affinity chromatography is a popular way to isolate glycoproteins. However, identifying glycosylated proteins that have been captured in this way can be difficult if the complexity of the sample is high. One standard solution to the general problem of sample complexity is to select a subset of tryptic peptides, thereby simplifying the identification step. In a new method that uses this strategy for glycoproteomics, Qiu et al., describe how they combined multiple affinity chromatography steps to narrow the search. First, they selected for proteins containing sugar moieties by lectin-affinity chromatography (specifically, a column containing Concanavalin A agarose beads). Then, Qiu et al., subjected the sample to proteolysis and strong anion exchange (SAX) and immobilized metal affinity chromatography (IMAC) columns. The SAX step is designed to select peptides with at least two acidic amino acids, whereas the copper-loaded IMAC column should further narrow the pool of acidic peptides to include only those that also have at least one histidine. These criteria do effectively limit the complexity of the peptide mixture that is subsequently used for MS-based peptide sequencing and parent glycoprotein identification, as shown by a pilot experiment with human transferrin. The authors also tested a real-world situation, namely the identification of glycoproteins from human serum. It is true that the Qiu et al., method missed 15 glycoproteins that had been described in a prior study which had used lectin affinity chromatography of the tryptic digest itself. However, some glycopeptides are not selectable by lectin and would therefore be missed by the previous method. Also, since a given tryptic digest would produce fewer glycopeptides than peptides containing acidic residues and histidine, the method proposed by Qiu et al., should return more peptide hits per protein, thereby improving the confidence of the identification.
- Qiu et al. 2006. A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides. Journal of Chromatography B [Epub ahead of print, August 29, 2006].
