Quantitative proteomics: a review of different methodologies.
The present review attempts to cover the vast array of methods which have appeared in the last few years for performing quantitative proteome analysis. These methods are divided into two classes: those applicable to conventional two-dimensional map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation [sodium dodecylsulfate (SDS)-electrophoresis] and those applicable to two-dimensional chromatographic protocols. The first method, although being by and large the most popular approach, can offer differential display of paired samples with relatively few methods, the oldest one being based on statistical analysis performed on sets of gels via powerful software packages, such as the MELANIE, PDQuest, Z3 and Z4000, Phoretix and Progenesis. Recent developments comprise analysis performed on a single gel containing mixed samples differentially labeled, either with fluorophors (Cy3 and Cy5) or with d(0)/d(3) acrylamide. Conversely, chromatographic approaches, which mostly rely on analysis not of intact proteins but of their tryptic digests, offer a panoply of differential labeling protocols, most of which rely on stable isotope tagging. Essentially, all possible reactions have been described, such as those involving Lys, Asp, Glu, Cys residues, as well as a number of methods exploiting differential derivatization of amine and carboxyl groups generated during proteolysis. All such methods are described and evaluated.
Righetti
Pier Giorgio
PG
Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada Le Grazie No. 15, 37134 Verona, Italy. righetti@sci.univr.it
Campostrini
Natascia
N
Pascali
Jennifer
J
Hamdan
Mahmoud
M
Astner
Hubert
H
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*