The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor-

We have investigated the regulation of p27 kip1 , a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G 0 ) to a proliferative (G 1 ) state. The level of p27 kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27 kip1, as well as a transient increase in cyclin D1-associated p27 kip1 that later declines concomitantly with the loss of total p27 kip1 . Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27 kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27 kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G 0 . Synthesis of p27 kip1 as determined by incorporation of [ 35 S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27 kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27 kip1. Northern (RNA) analysis of p27 kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27 kip1 mRNA, suggesting that the PDGF-regulated decrease in p27 kip1 expression occurred through a translational mechanism.Under normal circumstances, proliferation of mammalian cells is a highly controlled process. This control is achieved, in part, by growth factors and cytokines that exert either mitogenic and/or antiproliferative effects in a cell-specific manner (1). Binding of growth-regulatory ligands results in the activation of signaling pathways that trigger direct alteration of specific metabolic pathways as well as immediate and delayed changes in gene expression (65). Mitogenic stimulation initiates a program of sequential synthesis of proteins, termed cyclins, that complex with and activate cyclin-dependent kinases (cdks) (9, 29-31, 39, 52, 54, 64, 67), enzymes that modulate key regulatory events leading to progression through and transitions between different stages of the cell cycle (7,13,20,21,47,55,64). The order of cyclin synthesis during cell cycle traverse has been examined in a variety of cells, including T cells, macrophages, epithelial cells, and fibroblasts (16,30,31,44,45,50,52). The D-type cyclins, which complex with cdk4 and cdk6, are the first cyclins synthesized during the cell cycle. They are detected in mid-G 1 and are believed to function as Rb ...

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.

Uveal malignant melanoma (UM) is the most frequent primary intraocular tumour in adult humans. Because the survival rate of patients with UM has changed little in the past few decades, a better understanding of the molecular events governing UM development and the identification of markers indicating the potential for metastasis at the time of diagnosis are necessary to design improved and more specific treatments. In this study, we investigated UM tumour development by comparing two recently established UM cultures with different invasion potential by twodimensional gel electrophoresis. Protein features expressed differentially were identified by mass spectrometric analysis. Potential markers were assayed in both cultures and in long-term established UM cell lines (UW-1, OCM-1, SP6.5 and 92.1) by Western blotting and their role in invasion analysed using Matrigel membranes. Comparative analysis revealed that UM cultures with lowand high-grade invasion potential differ in their cellular metabolism and, more interestingly, in several cancer-associated proteins, including those implicated in cell adhesion and migration, proliferation and various oncogenes. Our data indicate a correlation between MUC18 and HMG-1 expression and the invasiveness of UM cells. We also demonstrate the expression and secretion of DJ-1 oncoprotein by UM cells. We suggest a possible role for MUC18 and HMG-1 proteins in UM cell invasion. The secretion of DJ-1 by UM cells, and the ability to detect this protein in UM patients' sera implicate it as a potential noninvasive biomarker for this malignancy. ' 2006 Wiley-Liss, Inc.Key words: uveal melanoma; invasion; proteomics; MUC-18; DJ-1 Uveal malignant melanoma (UM) is the most frequent intraocular tumour in adult humans.1 Unlike cutaneous melanoma, uveal melanoma disseminates mainly through the blood stream and preferentially establishes metastases in the liver. Metastatic liver disease is the leading cause of death in uveal melanoma and can develop after a long disease-free interval, which suggests the presence of occult micrometastatic disease at the time the primary eye tumour is diagnosed and treated.2 Unfortunately, advances in eye cancer treatment have not paralleled those made in the management of other types of cancer, and the survival rate of patients with uveal melanoma has changed little in the past few decades.3 A better understanding of the molecular events governing uveal melanoma development and the identification of markers indicating the potential for metastasis at the time of diagnosis are necessary to design improved and more specific treatments. Various clinical and molecular prognostic factors have been suggested in uveal melanoma, but none has proved to be sufficiently useful or viable for routine clinical use. 4,5 Currently, there are many challenging technologies and approaches to identify tumour markers for prognostic and therapeutic purposes.6 Transcriptional studies alone are not sufficient, with several investigators reporting a poor correlation between mRNA and p...