Trends in Biotechnology
ReviewWhat place for polyacrylamide in proteomics?
Section snippets
Competing technologies
Currently, there are two emerging technological approaches that compete with or complement 2D-PAGE in proteomic analysis. The first approach involves global proteolytic digestion of a complex sample and then analysis of this complex peptide mixture by tandem mass spectrometry (MS–MS), usually via an electrospray ionization interface. The number of peptides that can be identified by MS–MS is increased by partial separation of the mixture using one or more iterative in-line chromatography steps 8.
Low-abundance proteins
Most proteins are of low abundance. For example, 50% of the protein content of yeast is the product of 100 genes 13. This means that the remaining protein is distributed over thousands of gene products. In numerical terms, if there are about one million molecules of the most abundant proteins in yeast, the great bulk of proteins are present in just hundreds to tens-of-thousands of molecules per cell. These low-abundance proteins, are considered to be some of the most important, including
Membrane proteins
Few membrane proteins are seen on 2D gels when conventional sample-preparation methods are used. Under such conditions, hydrophobic membrane proteins aggregate when they concentrate at their pIs and are not readily solubilized by sodium dodecyl sulfate (SDS) for entry into the second dimension. Rabilloud's group (CEA, Grenoble, France) and ours have shown that strong solubilizing agents greatly reduce this problem 20, 26. However, improved solubility conditions alone are not sufficient to
Alkaline proteins
Alkaline proteins have been particularly difficult to resolve on 2D gels for two reasons. First, the most common commercially available pH gradients, until recently, were pH 4–7 and pH 3–10 and these do not provide significant alkaline-protein resolution. Resolution of proteins in immobilized pH gradients (IPG) up to pH 12 has been demonstrated 27, 28, and as more alkaline pH range IPGs become commercially available, the problem of detecting alkaline proteins on proteome maps will be at least
Characterization of protein isoforms
The process of determining whether a protein is expressed in a particular proteome has become a relatively simple task with the automation of the ‘in-gel’ digest and subsequent identification of the resulting peptides with mass spectrometers. Today, most proteins are identified by either assigning them definitive protein attributes, such as peptide masses generated by MALDI–TOF–MS or the short amino acid sequences generated by tandem MS. The data generated by MALDI–TOF–MS and tandem MS has led
Concluding remarks
Our conclusion from a careful reading of the literature and our own experience, is that 2D gels have been widely criticized because they used to require considerable technical skill to prepare, the results were not easily reproduced, and not enough material could be purified for detailed analysis. Today, these issues are solved. Using pre-cast mini gels (format of microtiter plate size), highly reproducible gels with outstanding resolution can be achieved by an inexperienced technician within a
References (32)
- et al.
2D protein electrophoresis: can it be perfected?
Curr. Opin. Biotechnol.
(1999) Automated LC–LC–MS–MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses
J. Chromatogr. B Biomed. Sci. Appl.
(2001)Proteomics: the industrialization of protein chemistry
Curr. Opin. Biotechnol.
(2000)Protein and antibody arrays and their medical applications
J. Immunol. Methods
(2001)Codon bias and gene expression
FEBS Lett.
(1991)- et al.
2D or not 2D. Two-dimensional gel electrophoresis
Curr. Opin. Chem. Biol.
(2001) - et al.
A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis
Anal. Biochem.
(2000) Enrichment of low-copy-number gene products by hydrophobic interaction chromatography
J. Chromatogr. A
(1999)Characterization of the yeast transcriptome
Cell
(1997)Isoelectric focusing in immobilized pH gradients in the pH 10–11 range
J. Biochem. Biophys. Methods
(1987)