2D-PAGE

Pietrogrande, M.C., Marchetti, N., Dondi, F., Righetti, P.G. (2006). Decoding 2D-PAGE complex maps relevance to proteomics. J. Chromatogr. B 833, 51-62. 

This section reviews the most recent results obtained by applying the above-described methods to complex 2D separations. In particular, the case of 2D-PAGE... 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

The major advantage of 2D-PAGE is that it enables simultaneous separation of thousands of unknown proteins, first by charge using isoelectric focusing (IEF) and... 

The statistical degree of overlapping (SDO) and 2D autocovariance function (ACVF) methods have been applied to 2D-PAGE maps (Marchetti et al., 2004 Pietrogrande et al., 2002, 2003, 2005, 2006a Campostrini et al., 2005) the means for extracting information from the experimental data and their relevance to proteomics are discussed in the following. The procedures were validated on computer-simulated maps. Their applicability to real samples was tested on reference maps obtained from literature sources. Application to experimental maps is also discussed. 

Moreover, experimental reference maps of human tissues were studied p7 and Mr coordinates of identified spots were retrieved from the SWISS-2D-PAGE database, the values <3X = 0.009 pH and av = 0.0002 log Mr were assumed for spot dimension since they represent the standard case for experimental 2D-PAGE maps—normal sample loading of a tissue homogenate (ca. 1 mg total protein) and standard gel sizes (18 x 20 cm, IEF x SDS-PAGE). 

FIGURE 4.12 Experimental 2D-PAGE map of a neuroblastoma xenograft implanted in mice. The circles indicate the spots identified by MS analysis. Reproduced from Campostrini et al. (2005) with permission from Wiley-VCH Verlag GmbH. 

It must be emphasized that the availability of the SMO and 2D autocovariance function methods as two independent statistical procedures to estimate the same parameter, in, the number of proteins, is a helpful tool to verify the reliability of the results obtained. In the case of the 2D PAGE map of colorectal adenocarcinoma cell line (DL-1) an excellent agreement was found between the values obtained from the SMO method—m = 101 10 and m = 105 10—and the 2D autocovariance function procedure—m = 104 10 (Pietrogrande et al., 2006a). 

Demalte-Annessi, I., Sanchez, J.-C., Hoogland, C., Rouge, V., Binz, P.-A., Appel, R.D., Hochstrasser, D.F. (1999). Submitted to the SWISS-2D-PAGE database. 

Dondi, F., Bassi, A., Cavazzini, A., Pietrogrande, M.C. (1998). A quantitative theory of the statistical degree of peak overlapping in chromatography. Anal. Chem. 70, 766. Eckerskom, C., Strupat, K., Schleuder, D., Hochstrasser, D.F., Sanchez, J.-C., Lottspeich, F., Hillenkamp, F. (1997). Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting. Anal. Chem. 69, 2888. Expasy,http //www.expasy.ch. 

FIGURE 4.1 2D polyacrylamide gel electrophoresis (2D-PAGE) maps of protein mixtures. (See text for full caption.)... 

Kraljevic To study the 2D-PAGE and Based on Using this... 

Cerfain proteins were confirmed by Western blot analysis. The 2D PAGE analysis can resolve up to 2000 proteins. In this study, 820 spots were mapped to proteins but only 165 were used in the protein-to-gene analysis. Presumably these represent the highest fold expressed genes from the Ixmg tumors that were also visible on the gels by silver staining. 

Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE lEF followed by SDS-PAGE)... 

After finishing the 2D-PAGE, proteins are separated both by their isoelectric points (pi) and molar masses (Mr). 

The discontinuous Laemmli SDS-PAGE with polyacrylamide concentration gradient in the separating gel is mosdy used for second dimension in 2D-PAGE (cf Protocol 2.1.1). Of course, other electrophoretic systems are also applicable. 

---