Molecular Biotechnology. Bernard R. Glick

Therefore, proteins are further separated on the basis of differences in their molecular weights (MW) by electrophoresis, at a right angle to the first dimension, through a sodium dodecyl sulphate-polyacrylamide gel.

      Depending on the size of the two-dimensional polyacrylamide gel and the abundance of individual proteins, approximately 2,000 different proteins can be resolved. The pattern of spots is captured by densitometric scanning of the gel. Databases have been established with images of two-dimensional polyacrylamide gels from some different cell types, and software is available for detecting spots, matching patterns between gels, and quantifying the protein content of the spots. Proteins with either low or high molecular weights, those with highly acidic or basic isoelectric points (such as ribosomal proteins and histones), those that are found in cellular membranes, and those that are present in small amounts are not readily resolved by 2D PAGE.

      After separation, individual proteins are excised from the gel and the identity of the protein is determined, usually by mass spectrometry (MS). A mass spectrometer detects the masses of the ionized form of a molecule. For identification, the protein is first fragmented into peptides by digestion with a protease, such as trypsin, that cleaves at lysine or arginine residues (Fig. 2.50). The peptides are ionized and separated according to their mass-to-charge (m/z) ratio, and then the abundance and m/z ratios of the ions are measured. Several mass spectrometers are available that differ in the type of sample analyzed, the mode of ionization of the sample, the method for generating the electromagnetic field that separates and sorts the ions, and the method of detecting the different masses. Peptide masses are usually determined by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS. To determine the m/z value of each peptide fragment generated from an excised protein by MALDI-TOF MS, the peptides are ionized by mixing them with a matrix consisting of an organic acid and then using a laser to promote ionization. The ions are accelerated through a tube using a high-voltage current, and the time required to reach the ion detector is determined by their molecular mass, with lower-mass ions reaching the detector first.

      Figure 2.50 Peptide mass fingerprinting. A spot containing an unknown protein that was separated by 2D PAGE is excised from the gel and treated with trypsin. Purified trypsin peptides are separated by MALDI-TOF MS. The set of peptide masses from the unknown protein are used to search a database that contains the masses of tryptic peptides for every known sequenced protein and the best match is determined. The trypsin cleavage sites of known proteins are determined from the amino acid sequence and, consequently, the masses of the tryptic peptides are easy to calculate. Only some of the tryptic peptide masses for the unknown protein are listed in this example.

      To facilitate protein identification, computer algorithms have been developed for processing large amounts of MS data. Databases have been established that contain the masses of tryptic peptides for all known proteins. The databases are searched to identify a protein whose peptide masses match the values of the peptide masses of an unknown protein that were determined by MALDI-TOF MS (Fig. 2.50). This type of analysis is called peptide mass 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