We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.

Abstract-Matrix metalloproteinase (MMP)-dependent shedding of heparin-binding epidermal growth factor (HB-EGF) and subsequent activation of the EGF receptor (EGFR) in the cardiovasculature is emerging as a unique mechanism signaling growth effects of diverse G protein-coupled receptors (GPCRs). Among these GPCRs are adrenoceptors and angiotensin receptors that contribute to the pathogenesis of hypertension through their vasoconstrictive and growth effects. Focusing on ␣ 1b -adrenoceptors, we suggest here that MMP-dependent activation of the EGFR promotes vasoconstriction as well as growth. We identified MMP-7 as a major HB-EGF sheddase in rat mesenteric arteries and ␣ 1b -adrenoceptors, angiotensin receptors, and hypertension-stimulated MMP-7 activity. Adrenoceptors stimulated EGFR autophosphorylation in arteries, and this transactivation was opposed by the MMP-7 inhibitor GM6001 as well as MMP-7-specific antibodies. In isolated microperfused arteries, blockade of EGFR transactivation with inhibitors of the EGFR (AG1478 and PD153035), HB-EGF (CRM197 and neutralizing antibodies), or MMPs (doxycycline) inhibited adrenergic vasoconstriction. In spontaneously hypertensive rats but not in normotensive rats, the inhibition of MMPs with doxycycline (19.2 mg/d from week 7 until week 12) reduced systolic blood pressure and attenuated HB-EGF shedding in the mesenteric arteries. These findings suggest a previously unknown mechanism of vasoregulation whereby agonists of certain GPCRs (such as adrenoceptors and angiotensin receptors) activate MMPs (such as MMP-7) that shed EGFR ligands (such as HB-EGF), which then activate the EGFR, thereby promoting vasoconstriction as well as growth. Because this mechanism is triggered by agonists typically overexpressed in hypertension, its blockade may have therapeutic potential for simultaneously inhibiting pathological vasoconstriction and growth in hypertensive disorders. Key Words: matrix metalloproteinase Ⅲ heparin-binding epidermal growth factor Ⅲ epidermal growth factor receptor Ⅲ vasoconstriction Ⅲ hypertension T here is increasing evidence implicating matrix metalloproteinases (MMPs) and metalloproteinase disintegrins (ADAMs) in shedding of growth factors (eg, heparin-binding epidermal growth factor [HB-EGF]) and thereby transactivation of cognate growth factor receptors (eg, EGF receptor) in the development of hypertrophy associated with hypertension. [1][2][3][4][5][6][7] In the heart, one mechanism of hypertrophy is transactivation of the EGF receptor by G protein-coupled receptors (GPCRs), such as adrenoceptors, angiotensin, and endothelin receptors, whose agonists (catecholamines, angiotensin II, and endothelins) are typically overexpressed as well as being historically implicated in the initiation, progression, and development of hypertensive disorders. 4,8 -12 These GPCRs transactivate the EGF receptor (EGFR) in cardiomyocytes via a shared pathway, whereby ADAM 12 sheds membraneanchored HB-EGF, which then binds to the EGFR either directly or via an interaction with the...

Background-Excessive stimulation of Gq protein-coupled receptors by cognate vasoconstrictor agonists induces a variety of cardiovascular processes, including hypertension and hypertrophy. Here, we report that matrix metalloproteinase-7 (MMP-7) and a disintegrin and metalloproteinase-12 (ADAM-12) form a novel signaling axis in these processes. Methods and Results-In functional studies, we targeted MMP-7 in rodent models of acute, long-term, and spontaneous hypertension by 3 complementary approaches: (1) Pharmacological inhibition of activity, (2) expression knockdown (by antisense oligodeoxynucleotides and RNA interference), and (3) gene knockout. We observed that induction of acute hypertension by vasoconstrictors (ie, catecholamines, angiotensin II, and the nitric oxide synthase inhibitor N G -nitro-

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images. Molecular & Cellular Proteomics 4:1205-1209, 2005.Large scale proteomic analyses of experimental model organisms provide valuable resources to a broad range of investigators working on both general and specific aspects of cell function. Recently there has been renewed interest in the bacterium Escherichia coli as a proof-of-concept model for systems-based approaches (1-3). Here we describe a proteomic analysis of E. coli with an eye toward creating a resource of potential value to system approaches as well as problem-based approaches.E. coli is probably the best understood of the simple model organisms and the most amenable to experimental analysis. Its genome has been fully sequenced (4), and the availability of complete genome sequence data bases facilitates the proteomic analysis of E. coli using MS. The proteome of an E. coli cell is estimated to have 4,285 proteins (5) with pI values ranging from 3.38 to 13.0 and molecular masses between 1.59 to 248 kDa (6 -8). These proteins are distributed among four well defined subcellular compartments: 1) the cytosol (2,885 known and predicted species), 2) the inner membrane (670 known and predicted species), 3) the outer membrane (87 known and predicted species), and 4) the periplasm, which separates the two membranes (138 known and predicted species).Recent "gel-free" proteomic approaches have coupled orthogonal chromatographic approaches with MS/MS (9) where one or more peptide tags serve as proxies for the identity, state, and abundance of a given protein. In the present work, we used the conventional and established method of 2D 1 -PAGE (10) for protein separation and analysis based on several consi...