Harpers

28 / CHAPTER 4GENOMICS ENABLES PROTEINS TO BEIDENTIFIED FROM SMALL AMOUNTSOF SEQUENCE DATAPrimary structure analysis has been revolutionized bygenomics, the application of automated oligonucleotidesequencing and computerized data retrieval and analysisto sequence an organism’s entire genetic complement.The first genome sequenced was that of Haemophilusinfluenzae, in 1995. By mid 2001, the completegenome sequences for over 50 organisms had been determined.These include the human genome and thoseof several bacterial pathogens; the results and significanceof the Human Genome Project are discussed inChapter 54. Where genome sequence is known, thetask of determining a protein’s DNA-derived primarysequence is materially simplified. In essence, the secondhalf of the hybrid approach has already been completed.All that remains is to acquire sufficient informationto permit the open reading frame (ORF) thatencodes the protein to be retrieved from an Internetaccessiblegenome database and identified. In somecases, a segment of amino acid sequence only four orfive residues in length may be sufficient to identify thecorrect ORF.Computerized search algorithms assist the identificationof the gene encoding a given protein and clarifyuncertainties that arise from Edman sequencing andmass spectrometry. By exploiting computers to solvecomplex puzzles, the spectrum of information suitablefor identification of the ORF that encodes a particularpolypeptide is greatly expanded. In peptide mass profiling,for example, a peptide digest is introduced into themass spectrometer and the sizes of the peptides are determined.A computer is then used to find an ORFwhose predicted protein product would, if brokendown into peptides by the cleavage method selected,produce a set of peptides whose masses match those observedby mass spectrometry.PROTEOMICS & THE PROTEOMEThe Goal of Proteomics Is to Identify theEntire Complement of Proteins Elaboratedby a Cell Under Diverse ConditionsWhile the sequence of the human genome is known,the picture provided by genomics alone is both staticand incomplete. Proteomics aims to identify the entirecomplement of proteins elaborated by a cell under diverseconditions. As genes are switched on and off, proteinsare synthesized in particular cell types at specifictimes of growth or differentiation and in response toexternal stimuli. Muscle cells express proteins not expressedby neural cells, and the type of subunits presentin the hemoglobin tetramer undergo change pre- andpostpartum. Many proteins undergo posttranslationalmodifications during maturation into functionallycompetent forms or as a means of regulating their properties.Knowledge of the human genome therefore representsonly the beginning of the task of describing livingorganisms in molecular detail and understandingthe dynamics of processes such as growth, aging, anddisease. As the human body contains thousands of celltypes, each containing thousands of proteins, the proteome—theset of all the proteins expressed by an individualcell at a particular time—represents a movingtarget of formidable dimensions.Two-Dimensional Electrophoresis &Gene Array Chips Are Used to SurveyProtein ExpressionOne goal of proteomics is the identification of proteinswhose levels of expression correlate with medically significantevents. The presumption is that proteins whoseappearance or disappearance is associated with a specificphysiologic condition or disease will provide insightsinto root causes and mechanisms. Determination of theproteomes characteristic of each cell type requires theutmost efficiency in the isolation and identification ofindividual proteins. The contemporary approach utilizesrobotic automation to speed sample preparationand large two-dimensional gels to resolve cellular proteins.Individual polypeptides are then extracted andanalyzed by Edman sequencing or mass spectroscopy.While only about 1000 proteins can be resolved on asingle gel, two-dimensional electrophoresis has a majoradvantage in that it examines the proteins themselves.An alternative and complementary approach employsgene arrays, sometimes called DNA chips, to detect theexpression of the mRNAs which encode proteins.While changes in the expression of the mRNA encodinga protein do not necessarily reflect comparablechanges in the level of the corresponding protein, genearrays are more sensitive probes than two-dimensionalgels and thus can examine more gene products.Bioinformatics Assists Identificationof Protein FunctionsThe functions of a large proportion of the proteins encodedby the human genome are presently unknown.Recent advances in bioinformatics permit researchers tocompare amino acid sequences to discover clues to potentialproperties, physiologic roles, and mechanisms ofaction of proteins. Algorithms exploit the tendency ofnature to employ variations of a structural theme toperform similar functions in several proteins (eg, theRossmann nucleotide binding fold to bind NAD(P)H,