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PubMedDate not supplied

Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins.

The aim was to determine the proportion of membrane glycoproteins captured using concanavalin A or wheat germ agglutinin lectin affinity chromatography. Digests of the isolated proteins were separated by reversed-phase liquid chromatography and analyzed by matrix-assisted laser desorption tandem mass spectrometry. The two lectins identified different groups of proteins with a broad range of molecular mass and p/ values, including a number of proteins that overlapped the two groups. Approximately 30% of the proteins were positively identified as containing domains that were predicted using standard bioinformatics methods to be characteristic of integral membrane proteins. This approach represents an effective method of surveying the membrane protein pool of mammalian cells for subsequent proteomic analysis.

Cell Membrane↗
PubMed2004

Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Amino Acid Sequence↗
PubMed2007

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Affinity Labels↗
PubMedDate not supplied

Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures.

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.

Affinity Labels↗
PubMed2003

Affinity enrichment of plasma membrane for proteomics analysis.

Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.

Biotinylation↗
PubMed2004

Target class strategies in mass spectrometry-based proteomics.

The cornerstone of proteomics resides in using traditional methods of protein chemistry, to extract and resolve complex mixtures, in concert with the powerful engines of mass spectrometry to decipher peptide and protein identities. The broad utility of proteomics technologies to map protein interactions, understand regulatory mechanisms and identify biomarkers associated with disease states and drug treatments necessitates a targeted biochemical approach tailored to the characteristics of the tissue, fluid or cellular extract being studied. The application of affinity methods in proteomic studies to focus on particular classes of molecules is being used with increasing frequency and comprises the subject of this review. An overview of successfully applied affinity methods is provided, along with speculation on the use of innovative approaches. Sample preparation and processing are critical for proteomics with affinity reagents, as only functional and active proteins can be isolated in most cases. Considerations for methods of sample preparation to optimize affinity capture and release are also discussed.

Animals↗
PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗
PubMedDate not supplied

Enrichment of phosphoproteins for proteomic analysis using immobilized Fe(III)-affinity adsorption chromatography.

We described an efficient protocol to strongly enrich phosphoproteins from mixtures of total cellular proteins using homemade, recyclable Fe(III)-affinity columns. An integral feature of the method is the use of a detergent cocktail that allows use of different pHs for total protein extraction (pH 6.8) and for subsequent affinity capture of phosphoproteins (pH 3.4). Affinity captured proteins from rat fibroblasts were fractionated on 2D gels and random selection was identified by mass spectrometry. More than 85% of identified proteins were previously known to be phosphorylated. The specificity of the method was further validated by isolating proteins from (32)P labeled cells. Our comparison of the clusters of acidic residues in the captured proteins with acidic clusters in proteins of the rat genome indicates that affinity for phosphate groups dominates over adsorption of proteins with acidic clusters.

Adsorption↗
PubMed2007

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

Adenosine Triphosphatases↗
PubMed2005

An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network.

We developed an integrated proteomic approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26 S proteasome interaction network in yeast. We termed this approach QTAX for quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes. For this work, in vivo formaldehyde cross-linking was used to freeze both stable and transient interactions occurring in intact cells prior to lysis. To isolate cross-linked protein complexes with high purification efficiency under fully denaturing conditions, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification. Tandem affinity purification after in vivo cross-linking was combined with tandem mass spectrometry coupled with a quantitative SILAC (stable isotope labeling of amino acids in cell culture) strategy to carry out unambiguous protein identification and quantification of specific protein interactions. Using this method, we captured and identified the full composition of yeast 26 S proteasome complex as well as the two known ubiquitin receptors, Rad23 and Dsk2. Quantitative mass spectrometry analysis allowed us to distinguish specific proteasome-interacting proteins (PIPs) from background proteins and led to the identification of a total of 64 potential PIPs of which 42 are novel interactions. Among the 64 putative specific PIPs, there are ubiquitin pathway components, ubiquitinated substrates, chaperones, and transcription and translation regulators, demonstrating the efficacy of the developed approach in capturing in vivo protein interactions. The method offers an advanced technical approach to elucidate the dynamic protein interaction networks of the proteasome and can find a wide range of applications in the studies of other macromolecular protein complex interaction networks.

Amino Acid Sequence↗
PubMedDate not supplied

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.

Animals↗
PubMed2006

Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.

Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.

Amino Acid Sequence↗
PubMed2004

The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana.

Organelle proteomics is the analysis of the protein contents of a subcellular compartment. Proteins identified in subcellular proteomic studies can only be assigned to an organelle if there are no contaminants present in the sample preparation. As a result, the majority of plant organelle proteomic studies have focused on the chloroplast and mitochondria, which can be isolated relatively easily. However, the isolation of components of the endomembrane system is far more difficult due to their similar sizes and densities. For this reason, quantitative proteomics methods are being developed to enable the assignment of proteins to a specific component of the endomembrane system without the need to obtain pure organelles.

Arabidopsis↗
PubMed2006

[Advances in plant proteomics--I. Key techniques of proteome].

With the completion of genome sequences of the model plants, such as rice (Oryza sativa L.) and Arabidopsis thaliana, it has come into plant functional genomics era, which becomes a hard base of the appearance and development of plant proteomics. This review focuses on the background of proteomics, concept of proteomics and key techniques of proteomics. The key techniques of proteomics include separation, such as 2-DE (Two-Dimensional Electrophoresis), RP-HPLC (Reverse Phase High Performance Liquid Chromatography) and SELDI (Surface Enhanced Laser Desorption/ Ionization) protein chip, mass spectrometry, such as MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry) and ESI-MS/MS (Electrospray Ionization Mass Spectrometry/Mass Spectrometry), databases related to proteomics, quantitative proteome, TAP (Tandem Affinity Purification) and yeast two-hybrid system. Challenges and prospects of proteomic techniques are discussed.

Computational Biology↗
PubMed2002

Comparative proteomics based on stable isotope labeling and affinity selection.

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Animals↗
PubMed2004

Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Antibodies↗
PubMed2006

Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes.

In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.

Affinity Labels↗
PubMedDate not supplied

Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis.

A convenient synthesis of some homologous light isotope-coded affinity tags (ICAT-L) containing an acid-labile moiety between the affinity component biotin and an electrophilic polar linker is described. These light ICAT reagents give smooth mass spectral signals in tandem mass spectrometry (MS/MS) analyses of some commercially available cysteine-containing peptides. However, these ICAT molecules are designed for use in identification and relative quantification of whole or partially purified cellular and tissue proteomes. Since the biotin moiety can be readily cleaved off the reagent after mass tagging, undesired residual fragmentation patterns caused by biotin of derived peptides, as normally observed using biotin-containing ICAT reagents, are effectively eliminated. This strategy should enhance peptide sequence coverage significantly which, in turn, should result in improving the quality of data obtained during data-dependent peptide mass and tandem mass spectral analysis of whole proteomes.

Biotin↗