HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anticoagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multi- We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches.This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulinregulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.Glucose transport into mammalian muscle and fat cells is an important step in insulin action and is critical for the maintenance of glucose homeostasis within the body (1). In mammalian muscle and fat cells, insulin stimulation activates a phosphorylation cascade, which in turn causes intracellular vesicles that contain the glucose transporter GLUT4, 4 to translocate to the plasma membrane (PM) and fuse (2, 3). In the basal state GLUT4 is distributed between the endosomal system, the trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) compartment that is highly insulin-responsive (4 -6).The protein kinase Akt is activated in response to insulin and plays a critical role in GLUT4 translocation (1, 7). However, the link between the insulin signaling pathway and GLUT4 translocation is not fully understood. The insulin-dependent movement of GLUT4 vesicles to the PM is an Akt-independent process, and this is followed by an Aktdependent step likely involving the docking and fusion of vesicles with the PM (7-9). The mechanism by which Akt controls the docking and fusion of GLUT4 vesicles with the PM is not known. However, it was previously shown that a Rab GTPase-activating protein (RabGAP) known as AS160 is phosphorylated by Akt in response to insulin (10). How AS160 functions in GLUT4 trafficking and its cognate Rab proteins are not known. The role of a variety of Rab proteins in GLUT4 trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined (11-16). However, although these Rab proteins may participate in some aspects of GLUT4 trafficking, no compelling evidence for specific involvement in the insulin-regulated trafficking of GLUT4 has been found.In this study we describe four key findings that add to our understanding of GLUT4 traffic...

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

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