Biochemical and Molecular Approaches 304411 Protein detection methods

Perhaps the most common category of biomarkers is that of macromolecules (most typically, proteins or messenger RNA molecules) that can be measured in cells, cell extracts, or biological fluids. Advances made in biochemistry and molecular biology over the last few decades have greatly expanded the number and sensitivity of techniques that can measure specific macromolecules or panels of macromolecules. As knowledge of signal transduction pathways has expanded, so too have the tools with which to assay signaling events and disruptions thereof. Analysis of posttranslational modifications of cellular proteins is perhaps the most commonly applied approach to assessing the role of a potential drug target in mediating a given signaling process. The development of antibody reagents that can detect specific modifications in proteins has been critical in these efforts. This is exemplified by antibodies that can detect protein epitopes that have been phosphorylated on specific amino acid residues; a breakthrough was the development of an antibody specific for phosphotyrosine residues.3'4 This enabled immunoaffinity purification of many protein kinase substrates, and also indirect monitoring of protein kinase activity in cells via immunoblotting (Western blotting) techniques. When coupled with immunoprecipitation of specific kinase substrate proteins, assessment of phosphotyrosine abundance remains an effective method for determining kinase signaling activity, and is thus a useful biomarker technique for profiling targets that have kinase activity.5 Further refinements in antibody reagents have led to antibodies that can detect not only specific types of protein modifications but modifications in a specific epitope in a particular protein, thus eliminating the need for prior immunoprecipitation to interrogate a particular protein. Early examples of this are antibodies against phosphoserine sites in the retinoblastoma and p53 tumor suppressor proteins,6,7 and the list of site-specific phosphoserine and phosphotyrosine antibodies has been growing continuously. Some key practical considerations that should be taken into account with phospho-specific antibodies, such as potential differences between detection of endogenous and recombinant versions of proteins and specificity differences between polyclonal and monoclonal antibodies targeting the same phosphoepitope, are discussed by Craig et al.8 If specificity of detection can be clearly established for such site-specific antibodies via the use of appropriate positive and negative controls, they present a considerable advantage in efficiency over more laborious immunoprecipitation methods.

It should be noted that although antibodies are the primary tool in the measurement of specific proteins and protein epitopes, other types of specific protein capture agents have emerged in recent years. Examples include affibodies, which are small protein domain proteins, derived from combinatorial engineering of the Z domain of Staphylococcal protein A, that can be selected via phage display libraries and can bind with high affinity to specific targets9'10; and aptamers, which are synthetic single-stranded DNA or RNA molecules that fold into structures that bind to specific proteins with high affinity.11 When conjugated with a photoreactive 5-bromo-deoxyuridine moiety, aptamers become photoaptamers, which can be photoactivated to form covalent cross-links with target proteins. While affibodies and aptamers may present some advantage in terms of scalability, flexibility in multiplexing applications (due to lower potential for cross-reactivity), and perhaps enhanced binding affinity, it remains to be determined whether or not either or these types of capture agents will supplant antibodies as broadly applicable reagents for detection of specific proteins in complex biological matrices. Meanwhile, if measurement of protein-protein interactions (or disruption thereof) is the objective, application of surface plasmon resonance technology enables affinity-based monitoring of protein interactions without the need for any labeling or detection agents (as the method detects differences in refractive index at the surface of an immobilized capture molecule).12,13 Though perhaps most often employed in hit-to-lead screening or screening of antibody interactions, biosensor chips coupled with mass spectrometry can be generated that may become useful in characterizing interactions of novel targets or in biomarker discovery.14,15 Protein biomarkers of target modulation, signal transduction, and downstream biological effects

Changes in the abundance and specific modifications of proteins can provide useful information in two key areas: assessment of target modulation and characterization of the downstream effects of target activity or pharmacological modulation thereof. Target modulation is most readily characterized when pharmacological antagonism (or agonism) of a protein's activity results in changes in modifications of the protein itself; for example, in cases of receptor tyrosine kinase that exhibit autophosphorylation activity, where inhibition results in reduction in the amount of phosphorylated amino acid residues.5,16-18 Alternatively or in addition, assessment of target modulation can be made via the analysis of proteins that are part of the signaling pathway affected by the target protein (hence, 'downstream' of the point of intervention). In general, in order for a downstream molecule to be a reliable biomarker, its linkage to the target protein of interest should be solidly established, at least in the particular cellular model system wherein the target characterization and drug screening efforts will be focused. Thus, a protein that is, for example, a direct substrate of a kinase or phosphatase target of interest would be a useful biomarker, provided the availability of an assay to detect changes in phosphorylation status of the substrate. If a pathway is well characterized, for example a phosphorylation cascade such as that induced by epidermal growth factor receptor activation, then profiling of modifications to multiple downstream components (such as mitogen-activated protein kinase) is a reasonable approach19,20; this has the advantage of reducing the likelihood of a spurious finding due to technical or biological variability in a single endpoint, and thus provides greater confidence that a desired pharmacological effect is being triggered and gives a broader indication of the extent of the effect. Similarly to changes in protein modifications, downstream changes in the abundance of specific proteins, due to changes in the rate of turnover or of gene expression, can also be considered as potential biomarkers, as would disruption of protein complex formation; examples of these would be changes in secreted cytokine levels during inflammatory reactions or induction of receptor homo- or heterodimerization triggered by growth factor binding.21,22 Proteins that are well correlated with functional changes in cell physiology, such as the Ki67 nuclear antigen associated with cell proliferation or the caspase-3 fragments generated in cells undergoing apoptosis,19 represent biomarkers of biological consequences further downstream of signaling cascades.

There are several format options for antibody-based assays that can be employed in the assessment of target modulation via protein modifications or downstream consequences. For in vitro characterizations in experiments of moderate throughput, immunoblotting is perhaps the most straightforward approach and, since it features electrophoretic resolution based on molecular weight, provides visual evidence of molecular weight variants or of cross-reactivity with other protein species. Measurements of protein abundance typically rely on enzyme-linked chemiluminscent readout although fluorescence-based detection can be done with suitable gel readers and appropriately labeled detection antibodies. However, quantitation of signal strength via immunoblotting is often less robust than that enabled by the enzyme-linked immunosorbent (ELISA) 'sandwich' assay format, and ELISA can confer greater specificity due to the option of utilizing two distinct antibodies, the so-called 'capture' and 'detection' antibodies (although similar specificity can be gained in the immunoblotting approach by incorporating an immunoprecipitation step, sometimes referred to as 'pull-down,' of a given protein of interest prior to gel separation and membrane probing). The ELISA method, like the related enzyme immunometric assay (EIA) methods, is particularly useful in the analysis of levels of secreted or otherwise soluble factors that are present in fluid matrices (conditioned cell culture media, serum and plasma) and at low concentrations. Further, recent advances in multiplexing technology have led to the ability to measure dozens or even hundreds of different proteins simultaneously in the same sample; this is most commonly performed via antibody-tagged cytometric beads in solution or on planar antibody microarrays.24 Cytometric beads are versatile in that beads can be coded based either on color or size, and currently allow higher multiplexing of capture/detection antibodies within a single sample, due to lesser effect of antibody cross-reactivity issues as compared to planar sandwich arrays; a comparison of commercially available cytokine multiplex detection kits provides some practical guidance for assay selection and addresses comparison between individual ELISA assays and measurements of the same analyte when performed in multiplex.25 Another approach to antibody arrays, which can be applied in a highly multiplexed fashion, involves dual-labeling of two different biological samples in which differential protein abundance is being queried; proteins in each samples are directly tagged with, e.g., distinct fluorophores and incubated on the same antibody microarray.26 This approach eliminates the need for detection antibodies and the associated need to screen for cross-reactivity, but lacks the added specificity provided by the detection antibody and cannot easily discriminate whether a difference in signal is due to different amounts of a single protein or to the fact that the protein forms larger multiprotein complexes in one case (as all proteins in the complex would be tagged with fluorophore).

For detection of protein biomarkers in the full context of organs and tissues (in situ), histological approaches such as immunofluorescence or immunohistochemistry are typically utilized; these can provide indication as to which specific cells or structures in a given tissue specimen are expressing a particular protein species of interest, albeit generally with more limited quantitative rigor than immunoblot or ELISA methods (histological methods are typically most useful in later stages of target validation or in drug development19'27). Another option is to use flow cytometry methods to measure protein biomarkers in individual cells that are grown in suspension culture or that have been disaggregated from solid substrates or tissues.28 Also, it should be noted that nonantibody-based methods are emerging for kinetic analysis of proteins and protein modifications in cells; for instance, quantitative mass spectrometry approaches have been developed which rely upon stable isotope-labeled peptide standards for the monitoring of phosphorylation changes in specific proteins from cell lysates.29,30 The choice of which of these detection methods are applied will depend on many factors such as the type of specimen (e.g., cultured cells, tissue, biological fluids) and collection method (e.g., fresh or flash-frozen versus formalin fixed); degree of sensitivity and quantitation required; and specificity of antibodies (this can be more of a concern in in situ assays or flow cytometry, which lack the electrophoretic mobility information that is captured in gel-based assays or the dual capture/detection antibodies that are utilized in ELISAs). For any of the commonly used protein detection methods, a paramount concern is that the method of choice in a specific evaluation must be capable of reliably detecting relative differences between treatment groups or between longitudinal samples collected in the same background; this is particularly the case for discovery-oriented experiments, whereas absolute measurements can assume greater importance in clinical studies, depending on the questions addressed and the nature of assays being employed.

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