Subcellular analysis fractions

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." ... 

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... 

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... 

Sialic acid is an important component of membrane glycoproteins, which has been described in several reviews (e.g. Cook and Stoddart 1973, Hughes 1976, Jeanloz and Codington 1976, Warren 1976, Glick and Flowers 1978, and many others). Analysis of-subcellular membrane fractions has demonstrated the presence of NeuSAc in all fractions of cultured cells and tissue extracts. 

Proteomics is a child of disparate parents the revolutionary advent of genomic sequencing and the evolutionary extension of mass spectrometry to permit the analysis of peptides. The fusion of these advances initially created a vision of full inventories of proteins in a biological unit, such as a cell, a subcellular fraction, or a physiological fluid. Good progress has been made toward this in some cases [11], but the focus is shifting from encyclopedic surveys toward an emphasis on quantitative... 

It has been suggested [6] that these unusual sterols, especially in those cases where these unusual sterols comprise the entire sterol content of the organisms, likely replace conventional sterols as cell-membrane components. Evidence for this comes from subcellular fractionation and subsequent analysis of two marine sponges [10]. The sterol composition of the membrane isolates was found to be identical to that of the intact sponge. Most common variation of the marine sterol is in the side-chain, situated deep in the lipophylic environment of the phospholipid bilayer. This suggests that unusual fatty acids might accompany the sterols, and indeed this is often the case [8]. 

Enriched subcellular compartments can be analyzed by MS/MS to determine their constituent proteins. One advantage of analysis of different cellular fractions is pre-analytical simplification that offers rewarding yields in dealing with the proteins identified in large-scale MS experiments. One of the major initiatives of the Human Proteome Organization (HUPO) is the comprehensive characterization of the complete subproteome of each cell type. 

In rats administered a single dose of C-2-hexanone at 200 mg/kg by gavage, tissue distribution was reported to be widespread with highest counts in the liver and blood. No quantitative data were given on tissue distribution (DiVincenzo et al. 1977). An analysis of subcellular distribution of the C label in liver, brain, and kidney tissue indicated highest counts were associated with the crude lipid fraction and protein, with some recovery in DNA, and little or none in RNA. 

Graham JM (1993) The identification of subcellular fractions from mammalian cells. In Graham J, Higgins J (eds.) Meth Molec Biol, vol. 19. Biomembrane protocols. 1. Isolation and analysis. Humana Press, To-towa, N.J., p 1... 

The metabolic study, considered separately, consists of treatment of the animal with the radiolabeled compound followed by chemical analysis of all metabolites formed in vivo and excreted via the lungs, kidneys, or bile. Although reactive intermediates are unlikely to be isolated, the chemical structure of the end products may provide vital clues to the nature of the intermediates involved in their formation. The use of tissue homogenates, subcellular fractions, and purified enzymes may serve to clarify events occurring during metabolic sequences leading to the end products. 

Peters, T.J. (1983) Subcellular fractionation and enzymatic analysis of tissue biopsy specimens. In Methods of Enzymatic Analysis (ed. Bergmeyer, H.U.), 3rd edn. Verlag Chemie, Weinheim. 

The combination of glucagon with a high concentration of a Ca2+-mobilizing hormone can reverse the loss of Ca2+ from liver cells [159] or lead to a large accumulation of Ca2+ in these cells [160]. This is particularly pronounced when the extracellular Ca2+ concentration is increased [160] (Fig. 12) and presumably reflects a predominance of the stimulation of Ca2+ influx [143-145] over the mobilization of intracellular Ca2+ stores [5] induced by the combined hormones. Analysis of the Ca2+ content of subcellular fractions indicates that the extra Ca2+ accumulated by the cells under these conditions is found principally in the mitochondria [160]. As expected, the cytosolic Ca2+ concentration in the presence of the hormone combination is much greater than seen with either hormone alone (P.F. Blackmore, unpublished observations) and reaches the level (> 600 nM) at which mitochondria begin to accumulate Ca2+. 

Fi/Fo ATPase or other highly abundant mitochondrial proteins, in addition to other unexpected proteins from other subcellular locations. Of course, it is likely that no one actually looked for such proteins in rafts before and with the unbiased nature of proteomics one expects (or even hopes ) to find components that had not been previously described. However, any analysis of a biochemical fraction can only be as good as the initial preparation of that fraction and in previous lipid raft studies the minimum standard one has to meet to claim a protein is in rafts is to demonstrate sensitivity to cholesterol disruption. 

Sample complexity reduction strategies are required to enable neuroscientists to address the issues mentioned above, and facilitate meaningful applications of proteomic analyses. The feasibility of protein enrichment by subcellular fractionation has been demonstrated through the analysis of the rat brain sub-proteomes of cytosolic, mitochondrial and microsomal fractions (Krapfenbauer et al. 2003). Another well-established subcellular fractionation technique, synaptosomal isolation, has recently... 

This chapter will describe in detail the procedure for Western blotting of polypeptides and proteins separated on a denaturing polyacrylamide gel system. This procedure is used routinely in our laboratory for the analysis of polypeptides from a variety of subcellular fractions of whole tissue and cell lines, and has evolved over a number of years in the hands of several people. Many different immunoblotting procedures are currendy available details of variadons from the I-protein-A method described here are given in the Notes secdon, as are brief amendments covering electrotransfer from two-dimensional and isoelectric focusing systems. A detailed descripdon of sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) is not appropriate for this chapter, and the reader is referred to vol. 1, Chapter 6, and refs. 9-14. For details of the producdon of polyclonal and monoclonal andsera, Chapters 1-6 in this vol. 

Subcellular fractionation, sucrose density gradients Allows isolation of specific cellular organelles for organelle proteome analysis difficult to obtain completely pure preparations... 

After intravenous treatment of rats with 1200 mg/kg/d CPH for 3 days, homogenates of the renal cortex were separated into subcellular fractions and their protein composition analyzed. The results of the SDS-gel electrophoresis of the renal cortical subtractions showed significant alterations of the polypeptide pattern in the microsomal fraction. The analysis of the polypeptide composition of the microsomal fraction indicated that paralleling to the depletion of cytochrome P-450 isoenzymes in the molecular weight range 50-53,000 was the induction of a polypeptide of molecular weight 44,000 [74]. 

Both in vitro and in vivo metabohsm has to be studied. Most of the results reviewed are from in vitro studies. The metabolites are generated by the use of recombinant enzymes, e.g., related to the cytochrome P450 (CYP) complex, by the use of subcellular fractions, e.g., microsomes, cytosols, or S9 fractions, or by the use of hepatocytes or liver slices. In the case of in vivo metabolism studies, the metabohtes are generated in living animals or humans, and analysis is performed in urine, plasma, bile, and/or faeces. 

An example of this approach was described by Carini et al. [7] in investigating the in vitro metabolism of an NO-releasing nonsteroidal anti-inflaimnatoiy drag (NCX 4016). Possible metabolites were postulated (Figure 10.1). An LC-MS system was developed for the separation of these components in extracts of various rat hver subcellular fractions (S9, microsomes, cytosol). LC-UV-DAD analysis of extracts after 90-min incubation revealed that only HBA, SA, and HBN were detected. In addition, an unknown metabolite was detected, which was identified as a glutathione conjugate at the benzyl carbon of HBA or HBN. Quantitative analysis was performed to study the kinetics of the biotransformation [7]. 

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