cDNA fragments from the 5′ end of transcripts due to the use of random hexamers, and by reads that map to more than one site in a genome due to the presence of repeated sequences. However, because each transcript is represented by many different reads, these biases are expected to have minimal effects on quantification of a transcript.
Proteomics
Proteins are the molecular machines of cells. They catalyze biochemical reactions, monitor the internal and external environments of the cell and mediate responses to perturbations, and make up the structural components of cells. Some proteins are present at more or less the same levels in all cells of a multicellular individual or a population of unicellular organisms under most conditions, for example, proteins that make up ribosomes or the cytoskeleton. The levels of other proteins differ among cells according to the cells’ functions or change in response to developmental or environmental cues. Thus, analysis of the proteins that are present under particular biological conditions can provide insight into the activities of a cell or tissue.
Proteomics is the comprehensive study of all the proteins of a cell, tissue, body fluid, or organism from a variety of perspectives, including structure, function, expression profiling, and protein−protein interactions. There are several advantages to studying the protein complement (proteome) of cells or tissues compared to other genomic approaches. Although analysis of genomic sequences can often identify protein coding sequences, in many cases the function of a protein, and the posttranslational modifications that influence protein activity and cellular localization, cannot be predicted from the sequence. On the other hand, it may be possible to infer a protein’s function by determining the conditions under which it is expressed and active. While expression profiles of protein coding sequences can be determined using transcriptomics, mRNA levels do not always correlate with protein levels and do not indicate the presence of active proteins, and interactions between proteins cannot be assessed by these methods. Generally, mRNA is turned over rapidly, and therefore, transcriptomics measures actively transcribed genes, whereas proteomics monitors relatively more stable proteins. From a practical standpoint, proteomics can be used to identify proteins associated with a clinical disorder (protein biomarkers), especially in the early stages of disease development, that can aid in disease diagnosis or provide targets for treatment of disease.
Identification of Proteins
A cell produces a large number of different proteins that must first be separated in order to identify individual components of the proteome. To reduce the complexity, proteins are sometimes extracted from particular subcellular locations such as the cell membrane, nucleus, Golgi apparatus, endosomes, or mitochondria. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is an effective method to separate proteins in a population (Fig. 2.49A). Proteins in a sample are first separated on the basis of their net charge by electrophoresis through an immobilized pH gradient in one dimension (the first dimension) (Fig. 2.49A). Some amino acids in a polypeptide have side chains with ionizable groups that contribute to the net charge of a protein; the degree of ionization (protonation) is influenced by the pH of the solution. In a gel to which an electric current is applied, proteins migrate through a pH gradient until they reach a specific pH (the isoelectric point) where the overall charge of the protein is zero and they no longer move. A particular position in the pH gradient may be occupied by two or more proteins that have the same isoelectric point. However, the proteins often have different molecular weights and can be further separated according to their molecular mass by electrophoresis at right angles to the first dimension (the second dimension) through a sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 2.49B). The separated proteins form an array of spots in the gel that is visualized using Coomassie blue, silver, or fluorescent protein stains.
Figure 2.49 2D PAGE for separation of proteins. (A) First dimension. Isoelectric focusing is performed to first separate proteins in a mixture on the basis of their net charge. The protein mixture is applied to a pH gradient gel. When an electric current is applied, proteins will migrate either toward the anode (+) or cathode (–) depending on their net charge. As proteins move through the pH gradient, they will gain or lose protons until they reach a point in the gel where their net charge is zero. The pH in this position of the gel is known as the isoelectric point and is characteristic of a given protein. At that point, a protein no longer moves in the electric current. (B) Second dimension. Several proteins in a sample may have the same isoelectric point and therefore migrate to the same position in the gel in the first dimension. 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
