title: Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches.
abstract: The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.
authors.affiliation: Department of Agricultural and Industrial Biotechnologies, University of Verona, Verona, Italy. righetti@sci.univr.it
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
authors.forename: Annalisa
authors.initials: A
authors.lastname: Castagna
authors.forename: Francesca
authors.initials: F
authors.lastname: Antonucci
authors.forename: Chiara
authors.initials: C
authors.lastname: Piubelli
authors.forename: Daniela
authors.initials: D
authors.lastname: Cecconi
authors.forename: Natascia
authors.initials: N
authors.lastname: Campostrini
authors.forename: Paolo
authors.initials: P
authors.lastname: Antonioli
authors.forename: Hubert
authors.initials: H
authors.lastname: Astner
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Hamdan
mesh_terms: D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007554:Isotopes; D040901:Proteomics
chemical_list: D007554:Isotopes