Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well‐characterized “real life” sample that can be used as reference standard in urine clinical proteomics studies.Experimental design: We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE‐MS, LC‐MS, LC‐MS/MS, 1‐D gel analysis in combination with nano‐LC MS/MS (using LTQ‐FT ultra), and 2‐DE‐MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds.Results: The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in “inter‐laboratory” and “inter‐platform” data comparison is also demonstrated.Conclusions and clinical relevance: These well‐characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae ; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.