Growth condition‐dependent cell surface proteome analysis of Enterococcus faecium

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid‐log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was “shaved” using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI‐bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi‐resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 ( http://proteomecentral.proteomexchange.org/dataset/PXD002497 ).     

2‐D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild‐caught and hormone‐treated F1 fish

In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild‐caught males. To gain insights into the molecular mechanisms that may be altered in the F1 testis, we used 2‐D DIGE to compare the protein profiling of the testis of wild‐caught males at the spermiation stage with that of F1 males showing different stages of germ cell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins by MS/MS revealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germ cell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males.     

Protein 2DE reference map of the anterior midgut of the blood‐sucking bug Rhodnius prolixus

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano‐LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 ( http://proteomecentral.proteomexchange.org/dataset/PXD001488 , http://proteomecentral.proteomexchange.org/dataset/PXD001489 ).     

Proteomic analysis of N‐glycosylation of human seminal plasma

Seminal plasma is a mixture of secretions from several male accessory glands. The seminal plasma contains many secreted proteins which are important for sperm function and male fertility. In this study, we employed N‐linked glycosylated peptide enrichment, combined with LC–MS/MS analysis, and establish the first large scale N‐linked glycoproteome of human seminal plasma. Combined with the results of five biological replicates, a total of 720 N‐glycosylated sites on 372 proteins were identified. Analysis of variations among five individuals revealed similar compositions of N‐glycosylated proteins in seminal plasma. The N‐linked glycoproteome could help us understanding the biological functions of human seminal plasma. The data set could also be a resource for further screening of biomarkers for male diseases including cancer and infertility at the level of N‐glycosylation. For example, N‐glycosylated prostate‐specific antigen is known to be an efficient biomarker that can distinguish benign prostate hyperplasia from prostate cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD000959 ( http://proteomecentral.proteomexchange.org/dataset/PXD000959 ).     

Calibration plot for proteomics: A graphical tool to visually check the assumptions underlying FDR control in quantitative experiments

In MS‐based quantitative proteomics, the FDR control (i.e. the limitation of the number of proteins that are wrongly claimed as differentially abundant between several conditions) is a major postanalysis step. It is classically achieved thanks to a specific statistical procedure that computes the adjusted p‐values of the putative differentially abundant proteins. Unfortunately, such adjustment is conservative only if the p‐values are well‐calibrated; the false discovery control being spuriously underestimated otherwise. However, well‐calibration is a property that can be violated in some practical cases. To overcome this limitation, we propose a graphical method to straightforwardly and visually assess the p‐value well‐calibration, as well as the R codes to embed it in any pipeline. All MS data have been deposited in the ProteomeXchange with identifier PXD002370 ( http://proteomecentral.proteomexchange.org/dataset/PXD002370 ).     

In‐depth characterization of the tomato fruit pericarp proteome

Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off‐line high‐pH reverse‐phase phase chromatography in combination with LC‐MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple‐TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.     

Qualitative and quantitative analysis of the adult Drosophila melanogaster proteome

Drosophila melanogaster is one of the most widely used model organisms in life sciences. Mapping its proteome is of great significance for understanding the biological characteristics and tissue functions of this species. However, the comprehensive coverage of its proteome remains a challenge. Here, we describe a high‐coverage analysis of whole fly through a 1D gel electrophoresis and LC‐MS/MS approach. By combining the datasets of two types of SDS‐PAGE and two kinds of tagmata, the high‐coverage analysis resulted in the identification of 5262 genes, which correspond to 38.23% of the entire coding genes. Moreover, we found that the fly head and body have different molecular weight distributions of their proteomes when the proteins were resolved with SDS‐PAGE and image analysis of the stained gel. This phenomenon was further confirmed by both label‐free and isobaric tags for relative and absolute quantitation‐based quantitative approaches. The consistent results of the two different quantitation methods also demonstrated the stability and accuracy of the LC‐MS/MS platform. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD000454 and PXD000455 ( http://proteomecentral.proteomexchange.org/dataset/PXD000454 ; ( http://proteomecentral.proteomexchange.org/dataset/PXD000455 ).     

Characterization of the Proteome of Theobroma cacao Beans by Nano‐UHPLC‐ESI MS/MS

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano‐UHPLC‐ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species‐specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non‐specific databases confirmed that using a species‐specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.     

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: Relevance for Lyme vaccine design

Lyme borreliosis is the most important vector‐borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC‐MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild‐type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT‐PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 ( http://proteomecentral.proteomexchange.org/dataset/PXD000876 ).     

Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins

The present study reports a comparative proteome cataloging of a bovine mastitis and a human‐associated Staphylococcus epidermidis strain with a specific focus on surfome (cell‐wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC‐MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house‐keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy‐metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein‐ and DNA‐mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO₂ conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 ( http://proteomecentral.proteomexchange.org/dataset/PXD000404 ).     

Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice

The four highly homologous members of the C‐terminal EH domain‐containing (EHD) protein family (EHD1‐4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4^−/− mice confirmed EHD4 deletion. Ehd4^−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4^−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4^−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age‐dependent manner. We conclude that high levels of EHD1 in the adult Ehd4^−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein‐mediated endocytic recycling as an important process in germ cell development and testis function. genesis 48:328–342, 2010. © 2010 Wiley‐Liss, Inc.     

Mapping the testicular interstitial fluid proteome from normal rats

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low‐abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI‐MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.     

Proteomic identification of Listeria monocytogenes surface‐associated proteins

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano‐LC and online‐MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy‐four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface‐exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.     

Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin

Studies of the secretomes of microbes grown on insoluble substrates are important for the discovery of novel proteins involved in biomass conversion. However, data in literature and this study indicate that secretome samples tend to be contaminated with cytoplasmic proteins. We have examined the secretome of the Gram‐negative soil bacterium Cellvibrio japonicus using a simple plate‐based culturing technique that yields samples with high fractions (60–75%) of proteins that are predicted to be secreted. By combining this approach with label‐free quantification using the MaxLFQ algorithm, we have mapped and quantified proteins secreted by C. japonicus during growth on α‐ and β‐chitin. Hierarchical clustering of the detected protein quantities revealed groups of up‐regulated proteins that include all five putative C. japonicus chitinases as well as a chitin‐specific lytic polysaccharide monooxygenase (CjLPMO10A). A small set of secreted proteins were co‐regulated with known chitin‐specific enzymes, including several with unknown catalytic functions. These proteins provide interesting targets for further studies aimed at unraveling the enzymatic machineries used by C. japonicus for recalcitrant polysaccharide degradation. Studies of chitin degradation indicated that C. japonicus indeed produces an efficient chitinolytic enzyme cocktail. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002843 ( http://proteomecentral.proteomexchange.org/dataset/PXD002843 ).     

Gel‐free mass spectrometry analysis of Drosophila melanogaster heads

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under‐represented group in proteomics datasets. Herein, a bottom‐up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem‐mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter‐aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed‐phase (RP) LC at high pH in the first dimension and acidic RP‐LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 ( http://proteomecentral.proteomexchange.org/dataset/PXD001712 ).     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).