PAPER IN FOREFRONT Emilio Marengo Æ Elisa Robotti Æ Daniela Cecconi Mahmoud Hamdan Æ Aldo Scarpa Æ Pier Giorgio Righetti Identification of the regulatory proteins in human pancreatic cancers treated with Trichostatin A by 2D-PAGE maps and multivariate statistical analysis Received: 8 January 2004 / Revised: 26 May 2004 / Accepted: 26 May 2004 / Published online: 15 July 2004 Ó Springer-Verlag 2004 Abstract In this paper, principal component analysis (PCA) is applied to a spot quantity dataset comprising 435 spots detected in 18 samples belonging to two dif- ferent cell lines (Paca44 and T3M4) of control (un- treated) and drug-treated pancreatic ductal carcinoma cells. The aim of the study was the identification of the differences occurring between the proteomic patterns of the two investigated cell lines and the evaluation of the effect of the drug Trichostatin A on the protein content of the cells. PCA turned out to be a successful tool for the identification of the classes of samples present in the dataset. Moreover, the loadings analysis allowed the identification of the differentially expressed spots, which characterise each group of samples. The treatment of both the cell lines with Trichostatin A therefore showed an appreciable effect on the proteomic pattern of the treated samples. Identification of some of the most rel- evant spots was also performed by mass spectrometry. Keywords PCA Æ Chemometrics Æ Human pancreatic tumour Æ Trichostatin A Æ Protein identification Introduction Since each cell or biological fluid has a rich protein content (often comprising thousands of proteins of dif- ferent structure and size), an effective method for achieving their separation is necessary. In the field of proteomics [1, 2], the separation of proteins is usually achieved by two-dimensional (2D) electrophoresis, a very powerful tool which performs two successive elec- trophoretic runs: the first run (through a pH gradient) separates the proteins with respect to their isoelectric point, while the second run (through a porosity gradient or a highly sieving, constant concentration gel) separates them according to their molecular mass. This technique produces a two-dimensional map, a so-called 2D-PAGE (polyacrylamide gel electrophoresis), with the proteins appearing as spots spread all over the gel matrix. A 2D- PAGE map may thus be considered as a ‘‘snapshot’’ of the protein content of the investigated cell at a given point of its life cycle. In this new post-genomic and proteomic era, the investigation of the protein content of different cell types has become fundamental. In fact, the physiological state of a particular cell or tissue is related to its protein con- tent, and the onset of a particular disease may cause differences in the proteins contained in the pathological tissue: these differences may consist of changes in the relative abundance or even in the appearance/disap- pearance of some proteins [3–11]. The comparison of 2D- PAGE maps belonging to healthy subjects with samples belonging to individuals affected by any pathology thus becomes a fundamental tool for both diagnostic and prognostic purposes [3–11]. The 2D-PAGE technique is also widely applied in the field of drug development [12–15], especially for cancer: two-dimensional gel-elec- trophoresis may be used to investigate if the treatment with a particular drug has played the expected role on the protein content of the pathological cell and to evaluate which effect was produced (e.g. up- or down-regulation, appearance/disappearance of pathological chains). E. Marengo (&) Æ E. Robotti Department of Environmental and Life Sciences, University of Eastern Piedmont, Spalto Marengo 33, 15100 Alessandria, Italy E-mail: emilio.marengo@mfn.unipmn.it Tel.: +39-131-287428 Fax: +39-131-287416 D. Cecconi Æ P. G. Righetti Department of Industrial Biotechnologies, University of Verona, Strada le Grazie 15, 37134 Verona, Italy M. Hamdan Computational, Analytical and Structural Sciences, GlaxoSmithKline, Via Fleming 4, 37135 Verona, Italy A. Scarpa Department of Pathology, Section of Anatomical Pathology, University of Verona, 37134 Verona, Italy Anal Bioanal Chem (2004) 379: 992–1003 DOI 10.1007/s00216-004-2707-x