Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.
A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.
Humphery-Smith
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I
University of Sydney, Centre for Proteome Research and Gene-Product Mapping, National Innovation Centre, Eveleigh, Australia. reeves04@angis.su.oz.au
Cordwell
S J
SJ
Blackstock
W P
WP
D000818:Animals; D004247:DNA / Q000235:genetics*; D004586:Electrophoresis / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D016678:Genome; D049673:History, 20th Century; D006801:Humans; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D017421:Sequence Analysis / Q000266:history / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D011506:Proteins; D012313:RNA; D004247:DNA