Subcellular analysis

In spite of the reduction in complexity that can be achieved by cellular fractionation, an analytical separation is frequently required to separate one or more components from the cellular milieu. As evidenced throughout this book, capillary electrophoresis (CE) provides high resolution and separation efficiency, both of which are necessary for subcellular analysis. In addition, CE is advantageous because it requires very little sample volume, typically less than a nanoliter, and generally very little sample preparation. Hence, capillaries have been used to directly sample subcellular compartments within neurons, oocytes, and muscle tissue sections. They have also been used to analyze individual organelles from a single cell following on-column lysis." ... 

Taking advantage of these attributes, CE has the potential to benefit diverse fields, that study the vital cellular processes that are localized within specific subcellular compartments. In fact, subcellular analysis by CE has already proven useful in different areas such as neurochemistry cellular physiology gene therapy drug accumulation, metabolism and localization disease diagnosis " and proteomics. ... 

In general, the aim of subcellular analysis is to quantify an analyte within a specific subcellular compartment. Consequently, in most cases, an organelle fraction must be purified before analysis. Cellular fractionation, that is, isolation and purification of organelles, has been indispensable in the biochemical fields and, as evidenced in the literature, has been used pervasively. Since complete reviews can be found in the biochemical literature, we will only briefly describe the principles of cellular fractionation. 

Subcellular analysis by CE can be performed in three modes (i) an organelle If action can be dissolved or lysed and the analytes found in the fraction analyzed (ii) intact, isolated organelles can be separated and detected and (iii) analytes or organelles can be directly sampled from a single cell or tissue section. Each mode of analysis will be illustrated below. 

Proteomics research has benefited greatly from subcellular fractionation, because reducing the complexity of the entire proteome to smaller organelle proteomes makes it possible to separate and detect low abundance proteins. Furthermore, since the goal of proteomics is not only to learn protein sequences but also the locahzation and function of proteins, subcellular analysis is advantageous because it provides the subcellular localization. Indeed, the benefits of cellular fractionation before... 

The resulting electropherogram is displayed in Figure 20.7, which shows the separation of all eight molecules. The separation was performed in Tris buffer that contained sulfated-P-cyclodextrin and hydroxypropyl-P-cyclodextrin, which provided the enantiomeric selectivity. Metabolites were quantified from 0.41 to 0.89 p.M with relative standard deviations of 10%. The subcellular analysis shows the preferential production of (-)-(/ )-metabolites (Table 20.1). Compared with a previously reported high-performance liquid chromatography (HPLC) method, " the CE method allowed these researchers to quantify two more metabolites and required less time. 

The third approach to subcellular analysis involves using the separation capillary to sample sub-cellular compartments within single cells or from tissue sections. Capillaries are uniquely suited to provide not only high resolution and separation efficiency but also, due to their physical dimensions, sufficient spatial resolution to sample subcellular compartments. Most commonly, the capillary is used to sample the cytoplasm from single cells. However, the capillary can also be used to sample intact organelles from tissue sections and analyze organelles released from single cells. ... 

Many complex cellular processes are best smdied through the use of an appropriate cell model. Cell cultures provide a relatively stable and virtually unlimited supply of organelles for subcellular analysis. We will discuss organelle preparations that are commonly used in the author s laboratory... 

To separate analytes from a dissolved organelle fraction, a useful and popular mode of separation is MEKC. As applied to subcellular analysis, the MEKC buffer is often used to dissolve the organelles and acts to separate the components based on their hydrophobicity. A common buffer utilized for this purpose is 10 mM borate, 10 mM sodium dodecyl sulfate (SDS) pH 9.5 (BS buffer), which has been used to separate doxombicin and its metabolites from nuclear, mitochondrial, and cytosolic-enriched fractions.Here, separations were performed from each fraction by injecting a small... 

In the biological chemistry field, fusion proteins have recently shown more potential as organelle-specific labels. For subcellular analysis, fusion proteins can be expressed that contain both a fluorescent protein and a subcellular localization sequence. This combination results in organelle-specific labeling that retains the excellent photochemical properties of fluorescent proteins (e.g., intense fluorescence and photostability). We commonly use a commercially available plasmid that codes for a fusion protein of red fluorescent protein (DsRed2) and the mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase, which contains the neomycin/kanamycin resistance gene, as described below. 

UV absorbance is the most common detection method for any CE system, primarily because nearly all compounds absorb somewhere in the UV region of the spectrum, and also because fused-silica capillaries are transparent to UV radiation. Given these advantages, UV absorbance detectors from HPLC instruments were naturally adopted into CE systems, but a problem arises when these detectors are used for subcellular analysis subcellular analyses commonly quantify analytes that are below the LOD for UV absorbance. According to Beer s law, the sensitivity of a UV absorbance detector is related to the pathlength of the light. Therefore, UV absorbance has limited applicability to traditional CE separations due to the short path of the typical capillary diameters. 

Lastly, the most popular detection technique for subcellular analysis is LIF due to the extremely low LODs that can be attained. Mass LODs on the order of attomoles can be achieved with commercial CE-LIF instruments and custom-built instruments, in conjunction with sheath-flow cuvettes, can reach down to the yoctomole (10 " mole) scale. LIF detection thus allows the quantification of minute amounts of analyte, and is the only detection method that has been used to detect individual organelles. We will therefore describe off-column LIF detection in further detail. 

As shown throughout this chapter, CE is a very versatile and powerful separation technique that has proven useful in subcellular analyses. As in all applications of CE, a trend for subcellular analysis is increasing throughput by using microfluidic devices to reduce separation times. This was recently demonstrated by analyzing fluorescently labeled mitochondria, for the first time, on a microfluidic chip. The analysis allowed a fivefold reduction in analysis time, from 20 to 4 min. To realize the full potential of CE, advances in electrophoretic models must also be made. Models that can accurately predict electrophoretic mobility, as well as explain observed differences, will allow this information to be used more effectively. Finally, as the information that can be gleaned from an electrophoretic... 

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