Quantitative protein profiling using two-dimensional gel electrophoresis,isotope-coded affinity tag labeling,and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.     

High-throughput screening of single-chain antibodies using multiplexed flow cytometry

We have developed a screening method that has the potential to streamline the high-throughput analysis of affinity reagents for proteomic projects. By using multiplexed flow cytometry, we can simultaneously determine the relative expression levels, the identification of nonspecific binding, and the discrimination of fine specificities to generate a complete functional profile for each clone. The quality and quantity of data, combined with significant reductions in analysis time and antigen consumption, provide notable advantages over standard ELISA methods and yield much information in the primary screen which is usually only obtained in later screens. By combining high-throughput screening capabilities with multiplex technology, we have redefined the parameters for the initial identification of affinity reagents recovered from combinatorial libraries and removed a significant bottleneck in the generation of affinity reagents on a proteomic scale.     

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.     

A versatile lentiviral expression system to identify mammalian protein-protein interactions

Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.     

Identification of novel tumor antigens with patient-derived immune-selected antibodies

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions.     

Selection of DNA nanoparticles with preferential binding to aggregated protein target

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life''s own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.     

Affinity reagent resources for human proteome detection: initiatives and perspectives

Essential to the ambition of characterising fully the human proteome are systematic and comprehensive collections of specific affinity reagents directed against all human proteins, including splice variants and modifications. Although a large number of affinity reagents are available commercially, their quality is often questionable and only a fraction of the proteome is covered. In order for more targets to be examined, there is a need for broad availability of panels of affinity reagents, including binders against proteins of unknown functions. The most familiar affinity reagents are antibodies and their fragments, but engineered forms of protein scaffolds and nucleic acid aptamers with similar diversity and binding properties are becoming viable alternatives. Recent initiatives in Europe and the USA have been established to improve both the availability and quality of reagents for affinity proteomics, with the ultimate aim of creating standardised collections of well-validated binding molecules for proteome analysis. As well as coordinating affinity reagent production through existing resources and technology providers, these projects aim to benchmark key molecular entities, tools, and applications, and establish the bioinformatics framework and databases needed. The benefits of such reagent resources will be seen in basic research, medicine and the biotechnology and pharmaceutical industries.     

Validation of affinity reagents using antigen microarrays

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Engineered bromodomains to explore the acetylproteome

MS‐based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti‐acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine‐containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot‐to‐lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.     

Iodine scanning of a phenazine inhibitor of vacuolar sorting

Affinity reagents are often used to address the target identification problem in chemical genetics. The design of such reagents so that the linker does not occlude interactions with protein targets is an ongoing challenge. This work describes a systematic approach to synthesize derivatives of a bioactive that should avoid interference with binding to targets and be readily converted to affinity reagents.     

Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labeling probes

The membrane proteome plays a critical role in electron transport processes in Shewanella oneidensis MR-1, a bacterial organism that has great potential for bioremediation. Biotinylation of intact cells with subsequent affinity-enrichment has become a useful tool for characterization of the membrane proteome. As opposed to these commonly used, water-soluble commercial reagents, we here introduce a family of hydrophobic, cell-permeable affinity probes for extensive labeling and detection of membrane proteins. When applied to S. oneidensis cells, all three new chemical probes allowed identification of a substantial proportion of membrane proteins from total cell lysate without the use of specific membrane isolation method. From a total of 410 unique proteins identified, approximately 42% are cell envelope proteins that include outer membrane, periplasmic, and inner membrane proteins. This report demonstrates the first application of this intact cell biotinylation method to S. oneidensis and presents the results of many identified proteins that are involved in metal reduction processes. As a general labeling method, all chemical probes we introduced in this study can be extended to other organisms or cell types and will help expedite the characterization of membrane proteomes.     

Medium throughput cage state stability screen of conditions for the generation of gold nanoparticles encapsulated within a mini-ferritin

Methods for the generation of nanoparticles encapsulated within cage proteins, such as ferritins, provide particles with low polydispersities due to size constraint by the cage. The proteins can provide enhanced water solubility to enable biological applications and affinity and identification tags to facilitate delivery or the assembly of advanced materials. Many effective methods have been developed, however, they are often impeded by cage protein instability in the presence of reagents or conditions for formation of the nanoparticles. Although the stability of ferritin cage quaternary structure can be enhanced, application of ferritins to materials science remains limited by unpredictable behaviour. Recently, we reported a medium throughput technique to directly detect the ferritin cage state. Herein, we expand this strategy to screen conditions commonly used for the formation of gold nanoparticles. Not only do we report nanoparticle formation conditions that permit ferritin stability, we establish a general screening strategy based on protein cage stability that could be applied to other protein cages or for the generation of other types of particles.     

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of na?ve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.     

Reactive oligonucleotide derivatives as gene-targeted biologically active compounds and affinity probes

Development of efficient methods for synthesis of oligonucleotides and oligonucleotide analogs has opened up the possibility of designing a broad spectrum of affinity reagents for specific modification of nucleic acids and proteins. These affinity reagents are used for investigation of the topology of ribosomes and nucleic acid polymerases. Oligonucleotides and their analogs are already used for suppression of specific gene expression and for elucidation of the physiological role of their products. Oligonucleotide derivatives appear to offer considerable promise as potential gene-targeted drugs such as antivirals and specific inhibitors of oncogene expression.     

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.     

Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.     

Affinity as a tool in life science

The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.     

Affinity-capture reagents for protein arrays

The simultaneous identification and quantitative measurement of the production levels of thousands of different proteins in a biological specimen remains an unachieved goal of modern proteomic research. Advances in the development of microarray-based platforms for highly parallel detection of proteins have therefore received a considerable impulse during the last few years. Here, we review the existing reagents for affinity capture of protein targets, as well as the techniques used for their immobilization on solid supports and methods for the detection of binding events, underlining the problems and the opportunities in this continuously evolving research field.