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OBJECTIVES
After studying this chapter, you should be able to:
Cite three examples of posttranslational modifications that commonly occur during the maturation of a newly synthesized polypeptide.
Name four chromatographic methods commonly employed for the isolation of proteins from biologic materials.
Describe how electrophoresis in polyacrylamide gels can be used to determine the purity, subunit composition, relative mass, and isoelectric point of a protein.
Describe the basis on which quadrupole and time-of-flight (TOF) spectrometers determine molecular mass.
Compare the respective strengths and weaknesses of DNA cloning and mass spectrometry (MS) as tools for determining protein primary structure.
Explain what is meant by “the proteome” and cite examples of its potential significance.
Describe the advantages and limitations of gene chips as a tool for monitoring protein expression.
Outline three strategies for resolving individual proteins and peptides from complex biologic samples to facilitate their identification by MS.
Comment on the contributions of genomics, computer algorithms, and databases to the identification of the open reading frames (ORFs) that encode a given protein.
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BIOMEDICAL IMPORTANCE
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Proteins are physically and functionally complex macromolecules that perform multiple critically important roles. An internal protein network, the cytoskeleton (see Chapter 51) maintains a cell’s shape and physical integrity. Actin and myosin filaments form the contractile machinery of muscle (see Chapter 51). Hemoglobin transports oxygen (see Chapter 6), while circulating antibodies defend against foreign invaders (see Chapter 52). Enzymes catalyze reactions that generate energy, synthesize and degrade biomolecules, replicate and transcribe genes, process mRNAs, etc (see Chapter 7). Receptors enable cells to sense and respond to hormones and other extracellular cues (see Chapters 41 and 42). Proteins are subject to physical and functional changes that mirror the life cycle of the organisms in which they reside. A typical protein is “born” at translation (see Chapter 37), matures through posttranslational processing events such as selective proteolysis (see Chapters 9 and 37), alternates between working and resting states through the intervention of regulatory factors (see Chapter 9), ages through oxidation, deamidation, etc (see Chapter 58), and “dies” when degraded to its component amino acids (see Chapter 29). An important goal of molecular medicine is to identify biomarkers such as proteins and/or modifications to proteins whose presence, absence, or deficiency is associated with specific physiologic states or diseases (Figure 4–1).
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