Francesca Antonucci

Two-dimensional maps in soft immobilized pH gradient gels: A new approach to the proteome of the Third Millennium

Same major improvements in proteome analysis of cytosolic and membrane proteins by two‐dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)‐gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2–3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri‐n‐butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240–220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al. (Electrophoresis 2001, 22, 2046–2057).

The proteome: anno Domini 2002.

Abstract We present some current definitions related to functional and structural proteomics and the human proteome, and we review the following aspects of proteome analysis: Classical 2-D map analysis (isoelectric focusing (IEF) followed by SDS-PAGE); Quantitative proteomics (isotope-coded affinity tag (ICAT), fluorescent stains) and their use in e.g., tumor analysis and identification of new target proteins for drug development; Electrophoretic pre-fractionation (how to see the hidden proteome!); Multidimensional separations, such as: (a) coupled size-exclusion and reverse-phase (RP)-HPLC; (b) coupled ion-exchange and RP-HPLC; (c) coupled RPHPLC and RP-HPLC at 25/60 °C; (d) coupled RP-HPLC and capillary electrophoresis (CE); (e) metal affinity chromatography coupled with CE; Protein chips. Some general conclusions are drawn on proteome analysis and we end this review by trying to decode the glass ball of the aruspex and answer the question: “Quo vadis, proteome”?

New approach based on fuzzy logic and principal component analysis for the classification of two-dimensional maps in health and disease: Application to lymphomas

Two-dimensional (2D) electrophoresis is the most wide spread technique for the separation of proteins in biological systems. This technique produces 2D maps of high complexity, which creates difficulties in the comparison of different samples. The method proposed in this paper for the comparison of different 2D maps can be summarised in four steps: (a) digitalisation of the image; (b) fuzzyfication of the digitalised map in order to consider the variability of the two-dimensional electrophoretic separation; (c) decoding by principal component analysis of the previously obtained fuzzy maps, in order to reduce the system dimensionality; (d) classification analysis (linear discriminant analysis), in order to separate the samples contained in the dataset according to the classes present in said dataset. This method was applied to a dataset constituted by eight samples: four belonging to healthy human lymph-nodes and four deriving from non-Hodgkin lymphomas. The amount of fuzzyfication of the original map is governed by the sigma parameter. The larger the value, the more fuzzy theresulting transformed map. The effect of the fuzzyfication parameter was investigated, the optimal results being obtained for sigma = 1.75 and 2.25. Principal component analysis and linear discriminant analysis allowed the separation of the two classes of samples without any misclassification.

Proteomics and immunomapping of reactive lymph-node and lymphoma

In the present study we show that two‐dimensional (2‐D) maps together with immuno‐detection allow the precise identification of important leukocyte differentiation and tumor markers (e.g., CD3 and CD5), and important cell cycle regulatory molecules such as cyclin dependent kinases, notably CDK6. In addition, the comparative evaluation of molecular expression (e.g., CD5) in maps developed with normal and lymphoma samples can provide reproducible and precise information regarding the molecular expression in different cell populations. Accordingly, we could detect a much increased level of expression of CD5 in mantle cell lymphoma, up to ten times higher than in the control. In addition, CD5 in tumor tissues seems to be microheterogeneous as compared to normal samples.

Chapter 15 Electrophoresis of proteins and peptides

Publisher Summary This chapter focuses on those methods of the electrophoresis of proteins and peptides that are widely adopted in modern separation science. These techniques are divided into two categories: gel-based and free-solution methodologies. Gel-based techniques include (1) conventional isoelectric focusing (IEF) in soluble carrier ampholyte buffers, (2) IEF in immobilized pH gradients (IPG), (3) sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and (4) two-dimensional maps as engendered by an orthogonal combination of IEF and IPG. The free-solution method is the capillary zone electrophoresis (CZE), which is the only useful approach currently used for performing fast separations of proteins/peptides in a free solution, although size separations in capillaries filled with appropriate solutions of sieving polymers can be easily performed. The chapter focuses on models for predicting the migration of peptides and presents recent studies aimed at assessing the folding/unfolding/misfolding processes that proteins undergo during denaturation/renaturation cycles.