Application to 2D Separations

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 

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). 

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Consequently, several hidden quantities can be estimated on the basis of the SMO approach. The procedure based on Equation 4.13 can be simply extended even to 2D separations as described in Fig. 4.7. In practice, the 2D pattern, in terms of spot positions and abundances, is divided into several strips. Each strip is transformed into a ID line chromatogram and the procedure described in Fig. 4.7 is then applied. Equation 4.13 is employed to calculate the m value of each strip from which the total m value is obtained. Applications to this procedure will be reported in Section 4.5. At this point, the reader s attention is drawn to the fact that the procedure of transforming 2D strips into ID chromatograms (see Fig. 4.7) once more corresponds to the overlapping mechanisms described in Fig. 4.2 and has been evocated in comparing Fig. 4.4 with Fig. 4.3. In this way, if random structures (e.g., such as those marked in Fig. 4.1b) are present, their memory is lost and the 2D pattern is reduced to a Poissonian ID one. Therefore, the number of SCs can be correctly estimated, even if the 2D pattern was not Poissonian. 

The goal of this chapter is to review the recent significant advances achieved in the study of 2D maps (Marchetti et al., 2004 Pietrogrande et al., 2002,2003,2005,2006 a Campostrini et al., 2005). Fundamental aspects concerning the intimate structure of multicomponent mixtures and separations will be discussed in Section 4.2. Description of the methods recently developed by the present authors for characterizing the separation pattern complexity of a 2D multicomponent map will be presented in Sections 4.3 and 4.4. These methods allow one to describe complex 2D separations in terms of SC number (m), the detection of hidden homologous series, spot shape features and separation performance. For these reasons they are named as decoding methods. In Section 4.5, the most recent achievements derived from the application... 

In any 2D HPLC, it is important to attain certain degree of both the complementarity and the orthogonality between the two separation dimensions [255-257]. The so far most universal approach to 2D polymer HPLC assumes the partial or possibly full suppression of the molar mass effect in the first dimension of the separation so that the complex polymer is separated mainly or even exclusively according to its chemical structure. Selected coupled methods of polymer HPLC are to be applied to this purpose. In the second dimension of separation—it is usually SEC—the fractions from the first dimension are further discriminated according to their molecular size. Exceptionally, SEC can be used as the first dimension to separate complex polymer system according to the molecular size. This approach is applicable when the size of polymer species does not depend or only little depends on their second molecular characteristic, as it is the case of the stereoregular polymers... 

Figure 7.4 2D representation of discriminant analysis. The dotted line represents the discriminant function and the solid line represents a discriminant surface that separates the two classes of samples. (From Livingstone, D.J., Data Analysis for Chemists Applications to QSAR and Chemical Product Design, Oxford University Press, Oxford, 1995. Reproduced with permission of Oxford University Press.)... 

Stable metal complexes can be favorably formed when a bidentate metal-binding site is available, such as a- and -diketone moieties which are the tautomeric forms of a- and /3-ketoenols. Some /S-diketonate complexes of paramagnetic lanthanides such as Pr(III), Eu(III) and Yb(III) have been extensively utilized as paramagnetic shift reagents for structural assignment of molecules with complicated stereochemistry prior to 2D techniques in NMR spectroscopy. Their syntheses and application are discussed in separate chapters in this volume. The examples below provide some dynamic and structural basis for better understanding of metal enolates in biomolecules and biochemical processes. 

Supercritical Fluid Extraction This process generally involves the use of CO2 or light hydrocarbons to extract components from liquids or porous solids [Brunner, Gas Extraction An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes (Springer-Verlag, 1995) Brunner, ed.. Supercritical Fluids as Solvents and Reaction Media (Elsevier, 2004) and McHugh and Krukonis, Supercritical Fluid Extraction, 2d ed. (Butterworth-Heinemann, 1993)]. Supercritical fluid extraction differs from liquid-liquid or liquid-solid extraction in that the operation is carried out at high-pressure, supercritical (or near-supercritical) conditions where the extraction fluid exhibits... 

Although liquid chromatography techniques have become quite popular in the separation of peptides in complex protein digests, they are yet to make an impact for the separation of protein samples for proteome-wide applications. It is envisioned that in the future their application for protein separation will increase. Various combinations of reversed-phase (RP)-HPLC with ion-exchange, size-exclusion, chromato-focusing (CF), IEF, and capillary electrophoresis (CE) have emerged for 2D separation of complex mixtures of proteins and peptides. A recent addition in this field is the use of CF as the first dimension and RP-FIPLC as the second-dimension separation device.14 CF is a column-based liquid-phase separation technique, in which proteins are fractionated on the basis of differences in their p/values in a weak ion-exchange column. 

The 2D gel or Iso-Dalt is the most commonly used method in proteomics because of its relatively easy use, automation, high reproducibility, high resolution of proteins, and applicability to analysis by mass spectroscopy. Furthermore, protein bands separated by Iso-Dalt are readily amenable to Edman degradation or to the amino acid composition analysis. 

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