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Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest. Therefore, these methods must be able to differentiate many antibodies at once. In this chapter, we describe a simple and rapid approach to labeling antibodies with distinct epitope tags in a site-specific manner. This allows multiple antibodies, even from the same host species, to be uniquely identified and detected and offers a simple approach for spatial proteomic applications.

Hypothalamic AMPK plays a key role in the control of energy homeostasis by regulating energy intake and energy expenditure, particularly modulating brown adipose tissue (BAT) thermogenesis. The function of AMPK can be assayed by analyzing its phosphorylated protein levels in tissues, since AMPK is activated when it is phosphorylated at Thr-172. Here, we describe a method to obtain hypothalamic (nuclei-specific) protein extracts and the suitable conditions to assay AMPK phosphorylation by Western blotting.

A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.

Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.

Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column.

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems, but the relative contribution of Tyr phosphorylation to plant signal transduction has, until recently, remained an open question. One of the major issues hampering the analysis is the low abundance of Tyr phosphorylation and therefore underrepresentation in most mass spec-based proteomic studies. Here, we describe a working approach to selectively enrich Tyr-phosphorylated peptides from complex plant tissue samples. We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment.

Tyrosine kinases are mainly classified into two groups, as receptor tyrosine kinase (RTK) and non-receptor tyrosine kinase (NRTK). The RTK family of transmembrane ligand-binding proteins are important mediators of the signaling cascade and includes EGFR, PDGFR (platelet-derived growth factor receptors), FGFR (fibroblast growth factor receptor) and the IR (insulin receptor). RTKs comprise 59 members and their structure includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain possessing the tyrosine kinase activity. This chapter focuses on antibody arrays that are basically used to analyse phosphorylation and dephosphorylation of RTKs. Antibody arrays include well-characterized antibodies for profiling, changes in RTK expression, and comparison between normal, diseased, or treated samples.

Single amino acid mutations in receptor tyrosine kinases (RTKs) are known to cause receptor over-activation and disease. Here we present a detailed protocol for the quantification of the effect of mutations on RTK activation in mammalian cells. The activation measurements are based on Western blotting, and involve direct comparison of receptor phosphorylation under conditions that ensure identical expression of wild-type and mutant receptors.

Immunoblotting for phosphorylated forms of receptor tyrosine kinases (RTKs) has been the mainstay of investigations on RTK signaling for the past two decades. Despite the development of quantitative mass spectrometry, reverse-phase protein array, and multiplex technologies, immunoblotting with phospho-specific antibodies is still used in parallel with these technologies and remains a powerful, and reproducible, method for interrogating signaling networks involving RTKs.

Phosphorylation of G protein-coupled receptors (GPCRs) is one of the most prominent post-translation modifications mediated by agonist stimulation. This process has been shown to result not only in receptor desensitisation but also, via the recruitment of arrestin adaptor proteins, to promote receptor coupling to numerous signalling pathways. Furthermore, there is now a growing body of evidence suggesting that GPCRs may employ phosphorylation as a mechanism to regulate their cell-type-specific signalling, hence generating tissue-specific functions. These advances have resulted partly from improved methods used in the determination of phospho-acceptor sites on GPCRs and improved analysis of the consequences of phosphorylation. This chapter aims to describe the methods used in our laboratory for the investigation of site-specific phosphorylation of the M₃-muscarinic receptor. These methods could easily be applied in the study of other receptors.

Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers to reveal key regulatory events and phosphorylation-mediated processes in the cell and in whole organisms. We present an overview of sensitive and robust analytical methods for phosphopeptide analysis, including calcium phosphate precipitation and affinity enrichment methods such as IMAC and TiO(2). We then discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large-scale experiments for studies of phosphorylation-mediated protein regulation.

Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of each secondary antibody with multiple primary antibodies when doing either dual- or triple-labeling immunofluorescence, it is necessary to use primary antibodies raised in different host species such as mouse, rabbit, and goat. However, in many cases, suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the first target protein followed by detection with antiprimary secondary antibody conjugated to NorthernLights™ NL-637 tag (fluorescence in the far-red spectral region). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-493 tag (green fluorescence) and anti-DIG secondary antibody conjugated to a Rhodamine Red X™ tag (red fluorescence). This technique provides good spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have only primary antibodies raised in the same host species at their disposal.

The in situ expression levels and subcellular localization of molecules involved in signal transduction using specific antibodies can be useful for prognosis and diagnosis of human diseases such as cancer. In addition, it has the potential to be helpful in monitoring biologic response to targeted therapies. The increasing availability of such antibodies makes these studies feasible. However, compared to typical immunohistochemical stains in which stabile molecules such as cytokeratins are targeted, additional -validation may be required for signal transduction immunohistochemistry.

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state. These phospho-specific antibodies can thus be used to track the activity of a protein, not simply its level of expression. In this review, we will discuss both the design of the phosphopeptide immunogen and immunization. The affinity purification of the phospho-specific antibody as well as the methods most suitable for characterizing the phosphospecificity of the antibody will be described here. Taken together, these methods will cover the key procedures and protocols required to produce a phospho-specific antibody that works.

Although phospho-specific primary antibodies used in immunohistochemistry (IHC) are expected to detect phosphorylated proteins, in some cases these antibodies may also cross-react with nonphosphorylated proteins. Therefore, it is of ultimate importance to employ a control to determine that the staining pattern is specific. One of the frequently used controls in IHC is a so-called absorption control: phospho-specific primary antibodies are first incubated with a phospho-peptide immunogen to block antibody-binding sites, and this mixture is subsequently applied to tissue sections. If the antibody blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific, but if staining is obtained, the antibody is considered nonspecific. Unfortunately, bound peptide can dissociate from the antibody allowing unblocked antibody to bind to tissue targets, producing unwanted staining. We have developed a simple absorption-control protocol allowing for the efficient neutralization of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of antibody-peptide complex from the incubation solution, minimizing the risk of formation of unblocked antibodies capable of producing tissue staining.

Growth factor stimulation results in phosphorylation of histone H3 at ser 10 and this correlated with expression of immediate early genes suggesting that this phosphorylation is associated with transcriptional activation. Although Western immunoblot analysis allows the detection of protein modifications in histones, in order to determine the localization of histones during different phases of cell cycle or during treatment of cells with different drugs we have to use immunohistochemistry. The protocol described here allows the detection of phosphorylated histones in tissue-cultured cells and tissue sections by fluorescent or bright-field immunostaining analysis. Here we used a serine 10 specific P-histone H3 antibody to determine the localization of this phosphoprotein in an asynchronously growing H4 glioma cell line and brain sections. It has been shown that long-term potentiation (LTP) is associated with gene transcription, and histone acetylation plays a major role in LTP formation (Wood et al., Learn Mem 13:241-244, 2006; Wood et al., Hippocampus 15:610-621, 2005; Alarcon et al., Neuron 42:947-959, 2004; Korzus et al., Neuron 42:961-972, 2004). Stimulus-induced phosphorylation of histone H3 at serine 10 has also been implicated in hippocampal neurons and striatal neurons (Li et al., J Neurochem 90:1117-1131, 2004; Crosio et al., J Cell Sci 116:4905-4914, 2003). Co-staining with a cell-specific antibody will allow us to determine the type of cells that show activation of histone phosphorylation in the brain.

In view of the importance of information transfer mediated throughout the cell by recognition, phosphorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The process can be easily automated using robotic liquid handlers and is extremely rapid, sensitive, and economical. Optionally, the method can be upgraded to a higher throughput level using more powerful workstations with greater capacity, such as the Biomek FX, or any similar robotics capable of transfer-from-file logic to guide synthesis cycles.

Protein tyrosine phosphorylation plays a central role in cell-signaling and is a focus of biomedical studies and cancer therapy. However, it is still challenging to identify or characterize the coordinated changes of many candidate proteins of one particular pathway or multiple pathways simultaneously. Antibody array is a recently developed approach applied for differential analysis of multiple protein posttranslational modification events in mammalian cells. It is based on the highly specific recognition between the immobilized antibodies on the array and their specific target proteins in a high-throughput screening format. Here we have described in detail two methods for differential analysis of protein tyrosine phosphorylation in cells by (1) using a single fluorescent protein capture format on membrane array and (2) a competitive protein capture method on glass surface array.

The T cell antigen receptor (TCR) is a multi-protein complex composed of six different transmembrane subunits, which form complexes of various sizes on the surface of resting T cells. The stoichiometry of the smallest form was recently determined to be alphabetagammaepsilondeltaepsilonzetazeta, whereas that of the larger forms is unknown. The roles of the different forms and their ratios are poorly defined. Biochemical analyses to address these questions must focus on the detergent and the best native conditions to maintain the integrity of the complexes. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a high-resolution native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. Using this powerful approach, the size, subunit composition and the relative abundance of the different TCR forms can be studied. We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE.

Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzyme-treated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described.

The biological functions of mast cells are regulated by several protein kinases, including the tyrosine kinases Fyn, Lyn, Syk, and FAK and the serine/threonine kinases Akt and PKC alpha/beta. The mitogen-activated protein kinases extracellular signal-regulated kinases, JNK, and p38MAPK also play a significant role in the regulation of mast cell biological function. This chapter will detail recent advances in determining mitogen-activated protein kinase activation in single cells. These methods are applicable to studies of signal transduction in mast cells.