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 prote in samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequ ence information, mass spectrometry has a key role to play in achievin g HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spec trometers or Liquid chromatography (LC)-based analyses are well advanc ed, 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 prot eins is proposed by employing principally mass spectrometry in an hier archical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analys is 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 hybridisatio n assays and proteome analysis may herald in a new era of holistic cel lular 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 mutatio ns); reaction kinetics; the influence of individual gene-products on b iochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic a nd epigenetic phenomena governing cellular processes. Proteome analysi s 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 engi nes necessary to achieve large-scale proteome analysis are outlined, a long with trends towards miniaturisation, analyte concentration and pr otein 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 s eparating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing diff erential display, high density expression arrays, expressed sequence t ags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular bio logy.