fingerprinting.
Protein Expression Profiling
Several methods have been developed to quantitatively compare the proteomes among samples. Two-dimensional differential in-gel electrophoresis is very similar to 2D PAGE; however, rather than separating proteins from different samples on individual gels and then comparing the maps of separated protein, proteins from two different samples are differentially labeled and then separated on the same two-dimensional polyacrylamide gel (Fig. 2.51). Typically, proteins from each sample are labeled with different fluorescent dyes (e.g., Cy3 and Cy5, which have higher sensitivity than many other protein stains); the labeled samples are mixed and then run together in the same gel, which overcomes the variability between separate gel runs. The two dyes carry the same mass and charge, and therefore, a protein labeled with Cy3 migrates to the same position as the identical protein labeled with Cy5. The Cy3 and Cy5 protein patterns are visualized separately by fluorescent excitation. The images are compared, and any differences are recorded. In addition, the ratio of Cy3 to Cy5 fluorescence for each spot is determined to detect proteins that are either up- or downregulated. Unknown proteins are identified by MS.
Figure 2.51 Protein expression profiling using 2D differential in-gel electrophoresis. The proteins of two different proteomes are labeled with fluorescent dyes Cy3 and Cy5, respectively. The labeled proteins from the two samples are combined and separated by 2D PAGE. The gel is scanned for each fluorescent dye, and the relative levels of the two dyes in each protein spot are recorded. Each spot with an unknown protein is excised for identification by MS.
Another powerful technique for comparing protein populations among samples utilizes protein microarrays. Protein microarrays are similar to DNA microarrays; however, rather than arrays of oligonucleotides, protein microarrays consist of large numbers of proteins immobilized in a known position on a surface such as a glass slide in a manner that preserves the structure and function of the proteins. The proteins arrayed on the surface can be antibodies specific for a set of proteins in an organism, purified proteins that were expressed from a DNA or cDNA library, short synthetic peptides, or multiprotein samples from cell lysates or tissue specimens. The arrayed proteins are probed with samples that contain molecules that interact with the proteins. For example, the interacting molecules can be other proteins to detect protein−protein interactions, nucleic acid sequences to identify proteins that regulate gene expression by binding to DNA or RNA, substrates for specific enzymes, or small protein-binding compounds such as lipids or drugs.
Microarrays consisting of immobilized antibodies are used to detect and quantify proteins present in a complex sample. Antibodies directed against more than 1,800 human proteins have been isolated, characterized, and validated, and subsets of these that detect specific groups of proteins such as cell signaling proteins can be arrayed. To compare protein profiles in two different samples, for example, in normal and diseased tissues, proteins extracted from the two samples are labeled with two different fluorescent dyes (e.g., Cy3 and Cy5) and then applied to one antibody microarray (Fig. 2.52). Proteins present in the samples bind to their cognate antibodies, and after washing to remove unbound proteins, the antibody-bound proteins are detected with a fluorescence scanner. Interpretation of the fluorescent signals that represent the relative levels of specific proteins in the two samples on a protein microarray is very similar to analysis of a DNA microarray.
Figure 2.52 Protein expression profiling with an antibody microarray. Proteins extracted from two different samples are labeled with fluorescent dyes Cy3 and Cy5, respectively. The labeled proteins are mixed and incubated with an array of antibodies immobilized on a solid support. Proteins bound to their cognate antibodies are detected by measuring fluorescence, and the relative levels of specific proteins in each sample are determined.
To increase the sensitivity of the assay and therefore the detection of low-abundance proteins, or to detect a specific subpopulation of proteins, a “sandwich”-style assay is often employed (Fig. 2.53). In this case, unlabeled proteins in a sample are bound to an antibody microarray, and then a second, labeled antibody is applied. This approach has been used to determine whether particular posttranslational protein modifications such as phosphorylation of tyrosine or glycosylation are associated with specific diseases. Serum proteins are first captured by immobilized antibodies on a microarray. Then, an antiphosphotyrosine antibody is applied that binds only to tyrosine phosphorylated proteins (Fig. 2.53A). The antiphosphotyrosine antibody is tagged, for example, with a biotin molecule, and fluorescently labeled streptavidin, which binds specifically to biotin, is added to detect the phosphorylated protein. In a similar manner, glycosylated proteins can be detected with lectins (Fig. 2.53B). Lectins are plant glycoproteins that bind to specific carbohydrate moieties on the surface of proteins or cell membranes, and many different lectins with affinities for different glycosyl groups (glycans) are available.
Figure 2.53 Detection of post-translational modifications with antibody microarrays. (A) Detection of tyrosine phosphorylation. An antibody microarray (1) is incubated with a protein sample (2). Biotinylated antiphosphotyrosine antibody is added (3) and, for visualization, a streptavidin-fluorescent dye conjugate attaches to the biotin of the antiphosphotyrosine antibody (4). (B) Detection of glycan groups. An antibody microarray (1) is incubated with a protein sample (2). A biotinylated molecule (e.g., lectin) that binds to a specific glycan is added (3) and, for visualization, a streptavidin-fluorescent dye conjugate attaches to the biotin of the lectin (4).
In another type of microarray, purified proteins representing as many proteins of a proteome under study as possible are arrayed on a solid support and then probed with antibodies in serum samples collected from healthy (control) and diseased individuals. The purpose of these studies is to discover whether individuals produce antibodies that correlate with particular diseases or biological processes. For example, the differential expression of antibodies in serum samples from individuals with and without Alzheimer disease was tested using a microarray consisting of more than 9,000 unique human proteins (Fig. 2.54). After incubation of the serum samples with the protein microarray, bound antibodies were detected using a fluorescently labeled secondary antibody that interacts specifically with human antibodies. The screen resulted in the identification of 10 autoantibodies (i.e., directed against an individual’s own protein) that may be used as biomarkers to diagnose Alzheimer disease. Protein microarrays can also be used to identify proteins that interact with therapeutic drugs or other small molecules (Fig. 2.55). This can aid in determining the mechanism of action of a drug, for assessing responsiveness among various forms of a target protein (e.g., variants produced by different individuals), and for predicting undesirable side effects.
Figure 2.54 Identification of disease biomarkers with a human protein microarray. Serum samples are collected from diseased and healthy individuals and incubated with microarrays of purified human proteins. Serum autoantibodies bind to specific proteins on the microarray and are detected by applying a fluorescently labeled secondary antibody directed against human antibodies. Autoantibodies present in the serum from diseased individuals
