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Apoptosis (programmed cell death) is an active process that plays a critical role in multiple biologic processes from embryologic development, to lymphocyte development and selection, and homeostasis. The two major mechanisms of cell death are referred to as the intrinsic and extrinsic pathways. These pathways lead to a cascade of events that ultimately converge to the activation of an effector enzyme, caspase-3. Caspase-3 is a cysteine protease with aspartic specificity and a well-characterized effector of apoptosis or programmed cell death signaling. The pro-form of caspase-3 (p32 caspase-3) is sequestered as a zymogen, where upon proteolysis at a conserved DEVD sequence, is converted to the active (p17 caspase-3) enzyme capable of disassembling the cell. Cell death can become disregulated under various conditions and multiple disease states (e.g., viral infection, carcinogenesis, and metastasis). Sensitive and reproducible detection of active caspase-3 is critical to advance the understanding of cellular functions and multiple pathologies of various etiologies. Here, we provide two simple and reproducible methods to measure active caspase-3 in multiple cell types and conditions using a flow cytometric-based analysis. [ABSTRACT FROM AUTHOR]

Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures. [ABSTRACT FROM AUTHOR]

Gene expression analysis provides an insight into the unique and defining biomolecular characteristics of a given cell type. However, heterogeneous cellular compositions hinder gene analysis studies from most tissue samples. The laser microdissection (LMD) technique allows for the unambiguous isolation of a desired cell population. However, preserving RNA integrity can be challenging because of the deliberately limited amount of starting material, sometimes as little as a single cell. General laboratory procedures for reducing ribonuclease (RNase) activity, both in reagents and in the laboratory environment, are required for successful downstream RNA isolation and quantitation. Quality RNA can be extracted from sections made from flash-frozen and paraffin-embedded tissue. The standard histological stains such as hematoxylin and eosin (H&E), or toluidine blue, can provide visualization of the cells of interest. Following LMD, validation of RNA integrity should precede downstream analysis. [ABSTRACT FROM AUTHOR]

The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the pro-apoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the CIP[-9.5]From: Methods in Molecular Biology, vol. 414: Apoptosis and Cancer Edited by: G. Mor and A. B. Alvero © Humana Press Inc., Totowa, NJ biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway. [ABSTRACT FROM AUTHOR]

Caspases are central to the execution of apoptosis. Their proteolytic activity is responsible for the demise of cells in many physiological and pathological states. Great advances in understanding caspases have been made using recombinant caspase expression and enzymatic characterization. Assays to measure caspase activity in apoptotic cell extracts and the development of a reconstituted cell-free assay were also critical in establishing the hierarchy in the caspase activation cascade and comprehend how caspase-9 is activated by the apoptosome. More recently, new tools such as activity-based probes allowed us to detect caspase activation in their working environment providing readout of the system with minimal interference. This chapter describes some of the methods used by our group to study the activation mechanisms of caspases and their activity. [ABSTRACT FROM AUTHOR]

Here we describe a simple protocol that uses positively charged nylon membrane dot blot to profile miRNA expression. A library of 515 antisense oligodeoxynucleotides of human and mouse mature miRNAs was synthesized and spotted on GeneScreen Plus membrane using a dot-blot equipment. Total RNA or enriched small molecular weight RNAs (smwRNAs) were enzymatically radiolabeled by poly (A) polymerase and then hybridized to the nylon membrane oligo arrays. The spot signal intensity on the membrane was analyzed using phosphorimaging. This method offers a convenient and economic way to simultaneously detect the expression of hundreds of miRNAs. [ABSTRACT FROM AUTHOR]

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer (1-4). The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in diseases such as Parkinson's and Alzheimer's citech13:bib05. The importance of the proteasome for general cell homeostasis has been established, and the 2004 Nobel Prize for Chemistry honored the researchers that discovered the ubiquitin-proteasome pathway. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autoflorescence or fluorescent library compounds. To address these issues, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. We have developed homogeneous, bioluminescent assays for all three proteasome activities, the chymotrypsin-like, trypsin-like, and caspase-like, using purified proteasome. We have also applied this technology to a cellular assay using the substrate for the chymotrypsin-like activity in combination with a selective membrane permeabilization step (patent pending). The proteasome assays are designed in a simple "add and read" format and have been tested in 96- and 384-well plates. The bioluminescent, coupled-enzyme format enables sensitive and rapid protease assays ideal for inhibitor screening. [ABSTRACT FROM AUTHOR]

Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice not only is more cost efficient but provides more informational content about a compound or treatment. For instance, multiplexed caspase activity assays can help establish the kinetics and magnitude of initiator and effector caspase induction by candidate compounds or treatments. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization that may help correct for differences in cell number. [ABSTRACT FROM AUTHOR]

Caspase activity assays in multi-well plate formats represent powerful tools for understanding experimental modulation of the apoptotic response. These assays are configured to exploit functional, biochemical, and temporal differences in substrate specificity and selectivity, which are useful in defining the magnitude and mechanism of a compound or treatment effect. New advances in fluorescent and luminescent chemistries now enable single addition "add-mix-measure" determinations of caspase activity directly in the sample plate with unprecedented sensitivity. Unlike other more cumbersome or laborious techniques, caspase activity induction or inhibition measures are quantifiable and definitive. The highlighted techniques in this chapter are cost efficient and allow for the rapid exploration of thousands of combinations and conditions. [ABSTRACT FROM AUTHOR]

Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer's disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research. [ABSTRACT FROM AUTHOR]

Since the discovery of the key role of cytochrome C in the activation of caspase 9, intense interest has focused on the role of mitochondria in apoptosis/programmed cell death. Mitochondria undergo two major alterations during apoptosis. The first is the permeabilization of the outer mitochondrial membrane. This event is tightly regulated by members of the Bcl-2 family and involves the conformational change of pro-apoptotic family members such as Bax. Second, the electrochemical gradient that is normally present across the inner mitochondrial membrane is lost (membrane depolarization). This event is sometimes mediated by the permeability transition pore (PTP). The order in which these events occur and whether one causes the other has been hotly debated in the literature. Nonetheless, the majority of reports suggest that mitochondria outer membrane permeabilization (MOMP) precedes membrane depolarization. In this chapter, methods that examine membrane depolarization and the conformational change in Bax are described. [ABSTRACT FROM AUTHOR]

Apoptotic markers include proteins from the intrinsic and extrinsic pathways. These cascades include both pro-apoptotic and anti-apoptotic elements. The expression levels of these elements can be assessed by immunohistochemistry (IHC) and can indicate general trends in pro- versus anti-apoptotic tendencies of the cells. IHC is particularly useful when studying large cohorts of paraffin-embedded specimens. Advances in tissue microarray (TMA) technology have facilitated evaluation of large cohorts of specimens, as cores from hundreds of patients can be represented on a single glass slide and stained in a uniform fashion. In this chapter, we discuss construction and staining methods of TMAs and present examples of assessment of apoptotic marker expression in malignant and benign cells using a novel method of automated, quantitative analysis of in situ protein expression. [ABSTRACT FROM AUTHOR]

The immediate assessment of response to therapy is most beneficial to ovarian cancer patients. This study shows the correlation of drug-induced caspase activation determined by western blot analysis and by Caspase-Glo™ assay. Our findings demonstrate that the use of the Caspase-Glo™ assay allows a simple, fast, and sensitive alternative for the evaluation of in vitro response to chemotherapy. [ABSTRACT FROM AUTHOR]

The ChemoFx® Assay is an ex vivo assay designed to predict the sensitivity and resistance of a given patient's solid tumor to a variety of chemotherapy agents. A portion of a patient's solid tumor, as small as a core biopsy, is mechanically disaggregated and established in primary culture where malignant epithelial cells migrate out of tumor explants to form a monolayer. Cultures are verified as epithelial and exposed to increasing doses of selected chemotherapeutic agents. The number of live cells remaining post-treatment is enumerated microscopically using automated cell-counting software. The resultant cell counts in treated wells are compared with those in untreated control wells to generate a dose-response curve for each chemotherapeutic agent tested on a given patient specimen. Features of each dose-response curve are used to score a tumor's response to each ex vivo treatment as "responsive," "intermediate response," or "non-responsive." Collectively, these scores are used to assist an oncologist in making treatment decisions. [ABSTRACT FROM AUTHOR]

Tumors are heterogeneous structures that contain different cell populations. Laser capture microdissection (LCM) can be used to obtain pure cancer cells from fresh-frozen cancer tissue and the surrounded environment, thus providing an accurate snapshot of the tumor and its microenvironment in vivo. We describe a new approach to isolate pure cancer cell population and evaluate protein expression. The process includes immunocytochemistry, laser microdissection, and western blot analysis. Using this technique, we can detect proteins such as X-linked inhibitor of apoptosis protein (XIAP) and Fas ligand (FasL) with as little as 1000 cells. [ABSTRACT FROM AUTHOR]

A number of methods have been developed to identify the cells that undergo apoptosis by analyzing the morphological, biochemical, and molecular changes that take place during this universal biological process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit. [ABSTRACT FROM AUTHOR]

Interference with the innate apoptotic activity is a hallmark of neoplastic transformation and tumor formation. Modulation of the apoptotic cascade has been proposed as a new approach for the treatment of cancer. In this chapter, we discuss the role of apoptosis in ovarian cancer and the use of phenoxodiol as a model for the regulation of apoptosis and potential use as chemosensitizer for chemoresistant ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Carbohydrates represent a fundamental building unit of living organisms. Many contemporary results introduce these substances as medium with remarkable data storage capacity (glycocode) that is decoded by special receptor counterpartners, lectins. Animal so-called endogenous lectins are presented here as the biotinylated tools for normal lectin histochemistry in accompany with biotinylated (neo)glycoconjugates employed in reverse lectin histochemistry. Practical lesson how to employ these probes in cell/tissue labeling including multiple cell labeling at the single-cell level is also included. Position of glycocode and endogenous lectins in squamous epithelium biology under physiological condition and after the malignant transformation is shown as example of the employment of mentioned probes in research and diagnostics. [ABSTRACT FROM AUTHOR]

The biotin-avidin microplate assay is a sensitive method to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases (MTases). The methylation reaction is carried out in solution using [methyl-3H]-AdoMet. Afterwards, the oligonucleotides are immobilized on an avidin-coated microplate, where the incorporation of [3H]-labeled methyl groups into the DNA is stopped by addition of unlabeled AdoMet to the binding buffer. Separation of radioactively labeled DNA from unreacted AdoMet and enzyme is performed by washing steps. Subsequently, the radioactivity incorporated into the DNA is released by a nucleolytic digestion of the DNA. By liquid scintillation counting, the amount of DNA methylation can be determined. Advantages of the microplate assay are its high sensitivity which allows the detection of low amounts of DNA methylation, the efficient separation of reaction components resulting in a low background of radioactivity and a high accuracy (±10%) and reliability. Furthermore, the assay is very convenient, fast and well suited for automation. [ABSTRACT FROM AUTHOR]

Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin-protein bonds that are susceptible to cleavage in human plasma. Here, we describe the use of immunoglobulin G as a model protein for evaluation of biotin-protein bond stability and for the investigation of the mechanism of biotin release. We also describe the synthesis of a biotin-protein bond that is stable in human plasma and a method for evaluation of that stability. [ABSTRACT FROM AUTHOR]

Analytical methods characterizing the immunogenicity of antigens are useful for monitoring, characterizing and predicting antibody responses to therapeutic biologics or vaccines. Distinct Luminex® microspheres coupled with protein G, anti-human immunoglobulin (Ig)A, anti-human IgM and anti-human IgE were developed for the simultaneous capture of total IgG, IgA, IgM and IgE (IgGAME) antibodies from human or non-human primate serum. The fraction of antigen-specific antibodies captured on the beads was detected using biotinylated antigen/streptavidin-phycoerythrin. The method was demonstrated by isotyping antibodies directed against an anti-CD11a antibody therapeutic (RAPTIVA®/efalizumab) from the serum of a cynomolgus monkey hyper-immunized with RAPTIVA® over a 15-month period. The quantitative range of the antibody measurements, using 5 μl of sample, was determined to be 15 ng/ml to 50 μg/ml in 10% serum. By the use of any biotinylated antigen as a detector, this multiplexed isotyping assay can be broadly applied to human and non-human primate IgGAME immunogenicity studies. [ABSTRACT FROM AUTHOR]

A number of endogenously biotinylated proteins are found in both cytosol and mitochondria of mammalian cells from many tissues, including liver, spleen, pancreas, kidney, and intestine. Therefore, caution should be taken when using the biotin detection system. Endogenous biotin can interfere with staining systems that employ the use of biotin-avidin- or biotin-streptavidin-based detection systems and may therefore result in high, non-specific background staining. Here, we show that this endogenous biotin reactivity can be deliberately exploited and used as a specific mitochondrial marker in both light and electron microscopy as well as for identifying mitochondrial fractions on Western blot. [ABSTRACT FROM AUTHOR]

Posttranslational modifications of histones play an important role in the regulation of chromatin structure and, hence, gene regulation. Recently, we have identified a novel modification of histones: binding of the vitamin biotin to lysine residues in histones H2A, H3, and H4. Here, we describe a procedure to identify those amino acids that are targets for biotinylation in histones. Briefly, the following analytical sequence is used to identify biotinylation sites: (i) short peptides (<20 amino acids in length) are synthesized chemically; amino acid sequences in the peptides are based on the sequence in a given region of a given histone; (ii) peptides are incubated with biotinidase or holocarboxylase synthetase to conduct enzymatic biotinylation; and (iii) biotin in peptides are probed using streptavidin peroxidase. Amino acid substitutions (e.g., lysine-to-alanine substitutions) in synthetic peptides can be used to corroborate identification of biotinylation sites. [ABSTRACT FROM AUTHOR]

Immunodetection techniques are dependent on enzyme-protein conjugates for the visualization of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. However, treatment of certain tissues with ABC reagents alone may result in high background, which is indicative for the presence of endogenous biotin or biotinylated proteins. In goldfish and salamander retinal sections, we observed a distinct staining pattern, presumably through binding of avidin to endogenous biotin in Müller cells. These findings summon for caution in the application of detection systems based on biotinylated antibodies or biotinylated DNA probes. [ABSTRACT FROM AUTHOR]

The interaction between avidin and biotin or streptavidin and biotin forms the basis of several widely used immunohistochemical techniques. An assumption inherent to these techniques is that endogenous biotin is not present in the tissue in detectable quantities, as neither avidin nor streptavidin can discriminate between endogenous biotin and biotinylated antibodies. However, biotin is a known cofactor for numerous carboxylases required in oxidative metabolism, raising the possibility for potential false-positive results in many tissues. This issue has been appreciated in liver and kidney tissue, but has received very little attention in nervous tissue. To address this concern, we examined the distribution of biotin throughout the rat central nervous system using avidin- and streptavidin-based detection systems, as well as a monoclonal antibody raised against biotin. Significant levels of endogenous biotin were identified within specific neuronal types, particularly in neurons associated with the cerebellar motor system and the brainstem auditory system. Non-specific (non-biotin) interactions of avidin and streptavidin conjugates with rat brain tissue were further identified and were most pronounced in the lower brainstem. The binding of avidin- and streptavidin-conjugated markers to endogenous biotin and other non-specific interactions with neural tissue were overcome by several methods including the use of blocking kits, prolonged post-fixation of the tissue in paraformaldehyde, or omission of Triton X-100 from the working solution. Without these measures, a reliable estimate of immunolabel may only be achieved in certain brain regions with markers conjugated directly to secondary antibodies. [ABSTRACT FROM AUTHOR]

The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites. [ABSTRACT FROM AUTHOR]

Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 μM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 μM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 μg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution. [ABSTRACT FROM AUTHOR]

The strong interaction between streptavidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for the facile derivatization of a variety of molecules (in particular, proteins) with biotin have been introduced, in order to allow their efficient recovery, immobilization and detection with streptavidin-based reagents. However, when desired, the release of biotinylated proteins from the streptavidin-based reagents remains a major problem, due to the extraordinary stability of this complex. This chapter presents a protocol developed in our laboratory for the quantitative elution of biotinylated proteins from streptavidin sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method is shown by the recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment. [ABSTRACT FROM AUTHOR]

Avidin functional affinity electrophoresis (AFAEP) is a variational method of affinity electrophoresis. In this technique, avidin is immobilized within a small area of the gel matrix by interaction with acrylamide and/or polyacrylamide either directly or through bifunctional linker glutaraldehyde during polymerization. Analytes can be heated with Tris-glycine sodium dodecyl sulfate (SDS) sample buffer so that biotinylated peptides/proteins are negatively charged and migrate electrophoretically towards the cathode through the avidin zone regardless of their isoelectric point (pI) values. Alternatively, if the behavior of the biotinylated analytes is known, the SDS treatment is not required. The polarity of the electrodes is set such that biotinylated analytes migrate electrophoretically through the avidin zone. This technique can work with or without SDS in gel running buffer. The AFAEP method allows the capture and concentration of biotinylated peptides/proteins. The values of this technique stem from a combination of merits of polyacrylamide gel electrophoresis and affinity technology. [ABSTRACT FROM AUTHOR]

Biospecific interactions are used in many capturing and bioseparation steps. A typical situation is the coupling of a biospecific ligand to a chromatographic stationary phase for affinity chromatography. This approach has two possible drawbacks. The first is that a chromatographic column may be awkward to use in experimental setups; the second is related to the need to develop a dedicated coupling chemistry for any given affinity ligand. In affinity precipitation, the biospecific affinity ligand is instead linked to a stimuli-responsive molecule to yield a so-called affinity macroligand (AML). Upon stimulation, such molecules show abrupt yet reversible precipitation from aqueous solution. Capture by affinity precipitation just requires the addition of the stimuli-responsive AML to the raw target solution followed by selective precipitation of the formed affinity complex via the application of the stimulus. The need for the synthesis of a dedicated AML may be circumvented by the use of an avidin-activated stimuli-responsive precursor, to which any biotinylated affinity ligand can be securely linked via the well-known strong avidin-biotin interaction. [ABSTRACT FROM AUTHOR]

Numerous methods have been published for the detection of protein using avidin-biotin technology. Complications can arise using this system when the protein of interest is in extremely high or low abundance. The ability to successfully detect high- or low-abundance proteins is dependent on the detection system selected. The expression of endogenous biotinylated proteins in rat liver homogenate will be used to illustrate the methods utilized for the detection of high- and low-abundance proteins on a membrane. The advantages and disadvantages of enzymatic and direct fluorescence detection systems are discussed. [ABSTRACT FROM AUTHOR]

The knowledge of the localization of proteins to a particular subcellular structure or organelle is an important step towards assigning function to proteins predicted by genome-sequencing projects that have yet to be characterized. Moreover, the localization of novel proteins to organelles also enhances our understanding of the functions of organelles. Many organelles cannot be purified. In several cases where the degree of contamination by organelles with similar physical parameters to the organelle being studied has gone unchecked, this has lead to the mis-localization of proteins. Recently, several techniques have emerged, which depend on characterization of the distribution pattern of organelles partially separated using density centrifugation by quantitative proteomics approaches. Here, we discuss one of these approaches, the localization of organelle proteins by isotope tagging (LOPIT) where the distribution patterns of organelles are assessed by measuring the relative abundance of proteins between fractions along the length of density gradients using stable isotope-coded tags. The subcellular localizations of proteins can be determined by comparing their distributions to those of previously localized proteins by assuming that proteins that belong to the same organelle will cofractionate in density gradients. Analysis of distribution patterns can be achieved by employing multivariate statistical methods such as principal component analysis and partial least squares discriminate analysis. In this chapter, we focus on the use of the LOPIT technique in the assignment of membrane proteins to the plant Golgi apparatus and endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Genome-wide transcriptional profiling provides a rich source of data for the validation and annotation of organelle proteomic data. Organelle biogenesis is in most cases accompanied by upregulation of genes encoding organelle-specific proteins. Consequently, identification of genes whose expression correlates with organelle assembly leads to a candidate list that can be cross-checked with a preliminary organelle proteome. When proteins are found in the proteome and the corresponding genes are found to have organelle assembly-correlated expression, this greatly increases our confidence that those proteins are true components of the organelle and not contamination. Such an approach can be used to narrow down a preliminary proteomic data set and help us to focus on a smaller sub-set of proteins that are supported by transcriptomic cross-validation. [ABSTRACT FROM AUTHOR]

Organelle protein profiles have traditionally been analyzed by subcellular fractionation of a specific organelle followed by the identification of the protein components of specific fractions containing the target organelle(s) using mass spectrometry (MS). However, because of limited resolution of the available fractionation methods, it is often difficult to isolate and thus profile pure organelles. Furthermore, many proteins (e.g., secretory proteins) are often observed to dynamically shuttle between organelles. Therefore, the determination of their true cellular localization requires the concurrent analysis of multiple organelles from the same cell lysate. Here, we report an integrated experimental approach that simultaneously profiles multiple organelles. It is based on the subcellular fractionation of cell lysates by density gradient centrifugation, iTRAQ labeling, and MS analysis of the proteins in selected fractions and principal component analysis (PCA) of the resulting quantitative proteomic data. Quantitative signature patterns of several organelles, including the ribosome, mitochondria, proteasome, lysosome, endoplasmic reticulum (ER), and Golgi apparatus, have been acquired from a single multiplexed proteomic assay using iTRAQ reagents. Through comparison PCA, we compare organelle profiles from cells under different physiological conditions to investigate changes in organelle profiles between control and perturbed samples. Such quantitative proteomics-based subcellular profiling methods thus provide useful tools to dissect the organization of cellular proteins into functional units and to detect dynamic changes in their protein composition. [ABSTRACT FROM AUTHOR]

Organelles are biochemically distinct subcellular compartments that perform specific functions within a cell. These roles are regulated by the complement of proteins associated with each organelle. Thus, a comprehensive understanding of an organelle's proteome is necessary to elucidate the diverse roles of each organelle. Mass spectrometry combined with biochemical fractionation methods has enabled the proteomic characterization of several organelles. However, due to the poorly quantitative nature of mass spectrometry and the inability to generate pure fractions of an organelle, distinguishing bona fide components of the organelle from contaminants of the fraction is a significant challenge. We have addressed this limitation by adopting quantitative mass spectrometric approaches to identify proteins that enrich in a purified fraction of organelles relative to a crude or contaminating fraction. The methods for the analyses of the yeast peroxisome are described in detail; however, these concepts are generally applicable to the study of other organelles. [ABSTRACT FROM AUTHOR]

Proteomics provides a powerful tool to characterize the protein content of an organelle. However, identifications obtained through mass spectrometry and database searching only make sense if the organelle sample is not heavily cross-contaminated. Besides the proteomic analysis, which gives an overview of possible cross-contamination, biochemical methods can be used to assess sample purity. These methods use specific markers that are detected and measured. Here, we describe the use of immunological, enzymatic, lipid, and pigment markers that allow the purity of chloroplast envelope fractions to be estimated. [ABSTRACT FROM AUTHOR]

This chapter describes the purification of ribosomal particles from a mutant strain of Escherichia coli using sucrose gradients and the characterization of their protein composition by a combination of mass spectrometry (MS) techniques. The main objective is to identify the ribosomal proteins that are missing in an aberrant ribosomal particle corresponding to a defective large subunit. To address this question, the tryptic digests of the purified ribosomal particles are analyzed by the coupling between liquid chromatography and tandem MS. The presence or absence of a given ribosomal protein in the defective particle is determined by comparing the MS intensities of its identified tryptic peptides with that of the mature large subunit. These analyses also allow identification of proteins copurifying with the ribosomal particles. To detect low-mass proteins escaping identification by the above method, intact proteins are also analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and nano-ESI-QqTOF MS. [ABSTRACT FROM AUTHOR]

The 20S proteasome is a multicatalytic protein complex present in all eukaryotic cells. Associated to regulatory complexes, it plays a major role in cellular protein degradation and in the generation of Major Histocompatibility Complex (MHC) class I antigenic peptides. In mammalian cells, this symmetrical cylindrical complex is composed of two copies of 14 distinct subunits, three of which possess a proteolytic activity. The catalytic standard subunits can be replaced by immunosubunits to form the immunoproteasome, which possesses different proteolytic efficiencies. Both types of 20S proteasomes can be present in cells in varying distributions. The heterogeneity of 20S proteasome complexes in cells leads to different protein degradation patterns. The characterization of the subunit composition of 20S proteasomes in cells thus represents an important step in the understanding of the effect of the heterogeneity of proteasome complexes on their activity. This chapter describes the use of proteomic approaches to study the subunit composition of 20S proteasome complexes purified from human cells. An immunoaffinity purification method is presented. The separation of all 20S proteasome subunits by 2D gel electrophoresis and the subunit identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis and database search are then described. These methods are discussed with the study of 20S proteasomes purified from two human cancer cell lines. [ABSTRACT FROM AUTHOR]

Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades arranged in a pinwheel-shaped array. Centrioles are required for recruitment of pericentriolar material (PCM) during centrosome formation, and they act as basal bodies, which are necessary for the outgrowth of cilia and flagella. Despite being described over a hundred years ago, centrioles are still among the most enigmatic organelles in all of cell biology. To gain molecular insights into the function and assembly of centrioles, we sought to determine the composition of the centriole proteome. Here, we describe a method that allows for the isolation of virtually "naked" centrioles, with little to no obscuring PCM, from the green alga, Chlamydomonas. Proteomic analysis of this material provided evidence that multiple human disease gene products encode protein components of the centriole, including genes involved in Meckel syndrome and Oral-Facial-Digital syndrome. Isolated centrioles can be used in combination with a wide variety of biochemical assays in addition to being utilized as a source for proteomic analysis. [ABSTRACT FROM AUTHOR]

Pancreatic zymogen granules (ZGs) are specialized for digestive enzyme storage and regulated secretion in exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to identify the ZG membrane (ZGM) proteins from ZGs purified by Percoll gradient centrifugation. By combining multiple separation strategies including two-dimensional gel electrophoresis and two-dimensional HPLC with tandem mass spectrometry, we identified 101 proteins from purified ZGMs including a large number of proteins previously unknown on ZGMs. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomics strategy was used to measure the enrichment of intrinsic membrane proteins through the purification steps by labeling crude, KBr-, and Na$_{2}$CO$_{3}$-washed ZGMs with multiplexed isobaric tags (iTRAQ™), 114, 116, and 117, respectively. The proteins with 117:114 ratios greater than one correlated well with intrinsic membrane proteins that contain either known or predicted transmembrane domains. [ABSTRACT FROM AUTHOR]

Synaptic vesicles store and subsequently release neurotransmitters into the synaptic cleft thereby regulating chemical neurotransmission in the brain. Proteins present in synaptic vesicles vary greatly in structure and function and have been identified primarily by genetic knock-out analysis in C. elegans, Drosophila, and mice (1,2). However, knock-out methods are not useful for the identification of proteins when a detectable phenotype is not created. Further, certain knocked-out proteins have function(s) that could be compensated for by another protein or cause a lethal phenotype when deleted. Additionally, some transporters and enzymes that appear to copurify with synaptic vesicles have not been characterized and confirmed (3-5). We have determined the proteins associated with purified synaptic vesicles using 2-D polyacrylamide gel electrophoresis (PAGE) protein separation followed by identification by mass spectrometry (6). Some of the new proteins identified were evaluated by western blot and confocal immunofluorescence analysis. [ABSTRACT FROM AUTHOR]

This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins (essentially soluble lysosomal proteins) from cells in culture is induced by adding a weak base in the culture medium. Secreted proteins are ammonium sulfate precipitated, dialyzed, and loaded onto the immobilized MPR column. After specific elution and collection of the M6P proteins, these are resolved by either bidimensional or monodimensional gel electrophoresis (designated as 2-DE or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised from the 2-DE gel, or on discrete bands covering altogether the whole length of the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and protein identification is obtained, thanks to peptide mass fingerprints [provided by analysis of the digests by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences (provided by analysis of the digests by the coupling between liquid chromatography and tandem mass spectrometry, LC-MS/MS). [ABSTRACT FROM AUTHOR]

Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping membrane has received scant attention. Advances in mass spectrometry and proteomics have laid the necessary groundwork to facilitate investigation of membranes such as those of lysosomes, mitochondria, and other organelles to find novel proteins and novel functions. Pure lysosomes are a prerequisite, and we have successfully identified an abundance of membrane proteins from lysosomes of rat liver. Here, we describe two comparable and easy methods to isolate lysosomes from mouse or rat liver in sufficient quantities for proteomics studies. Also included is a comparison of the soluble, luminal proteins obtained from each of the two preparations separated by 2D immobilized pH gradient (IPG) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). [ABSTRACT FROM AUTHOR]

Gas-phase fractionation (GPF), defined as iterative mass spectrometric interrogations of a sample over multiple smaller mass-to-charge (m/z) ranges, enables the ions selected for collision-induced dissociation to come from a greater number of unique peptides compared to the ions selected from the wide mass range scan in automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. GPF is described as a means to achieve higher proteome coverage than multiple LC-MS/MS analyses of unfractionated complex peptide mixtures. It is applied to organellar proteomics through analysis of yeast peroxisomal proteins obtained from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins. [ABSTRACT FROM AUTHOR]

Peroxisomes take part in various metabolic pathways related to the regulation of lipid homeostasis. Although detailed information on the enzymes involved in the peroxisomal lipid metabolism was acquired in the past, the mechanisms of metabolic exchange between peroxisomes and the cytosol or other organelles still remain an enigma. Therefore, a detailed analysis of the peroxisomal membrane proteome could help identify potential metabolite transporters. However, because of their highly hydrophobic character, membrane proteins tend to precipitate in aqueous media, making their fractionation still a challenging task. To overcome these obstacles, we have elaborated a protocol for the separation of both hydrophilic as well as hydrophobic proteins using free flow isoelectric focusing (FF-IEF). Similar to traditional gel-based isoelectric focusing, a denaturing electrophoresis buffer containing a mixture of urea, thiourea and detergents is applied to keep highly hydrophobic proteins in solution. Electrophoresis is conducted on a BD Free Flow Electrophoresis System with a linear pH gradient from 3 to 10 and sampled into 96 fractions. As a second dimension, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is used to further separate and visualize the protein pattern of the peroxisomal subfractions of matrix, peripheral and integral membrane proteins. The identification of the known peroxisomal membrane proteins PMP22, PMP70 as well as mGST in the subsequent matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of the 100 most prominent protein bands has documented the suitability of this new technique for the analysis of hydrophobic proteins. [ABSTRACT FROM AUTHOR]

Integral membrane proteins are represented by 20-30% of the eukaryotic genome and crucial for cellular functions including cell signaling, nutrient influx, toxin efflux, and maintenance of osmotic balance. Importantly, over 70% of all drugs are targeted at membrane proteins. Because of their hydrophobicity, however, methods used to characterize the structure of soluble proteins, such as NMR and X-ray crystallography, are generally not suitable to the study of membrane proteins (1). The methods described in this chapter facilitate the identification and mapping of both extracellular and cytoplasmicsoluble domains of integral plasma membrane proteins using mass spectrometry. By combining a classical protease protection approach with recently developed proteomic methods, protease-accessible peptides (PAPs) are digested from proteins embedded in their native lipid environment and identified to characterize the topologies of integral membrane proteins. [ABSTRACT FROM AUTHOR]

This chapter presents procedures for preparation and analysis of fractions enriched in plasma membranes from frozen tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The method for isolation of membranes comprises a stepwise depletion of non-integral membrane proteins from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin and enrichment of the plasma membranes by a density gradient fractionation. Reduction, carboxymethylation, and digestion with endoproteinase Lys-C are carried out on non-solubilized membranes. The entire procedure allows processing and preparation of samples of minute amounts as 10-20 mg tissue and therefore can be extremely helpful for proteomic profiling of small pieces of tissue and clinical material. [ABSTRACT FROM AUTHOR]