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In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiOsub2/subin a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.

The newborn screening for cystic fibrosis (CF) can identify affected but still asymptomatic infants. The selection of omic technique for gut microbiota study is therefore crucial due to both the small amount of feces available and the low microorganism load. Our aim was to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis including McNemar (taxa presence) test and Bland–Altman (taxa abundance) data plotting for studying the sequential establishment of the gut microbiota during the first year of life in 8 infants with CF (28 fecal samples). The taxonomic assignation was similar by the two techniques, whereas certain discrepancies were observed in the abundance detection, mostly the underrepresentation of Bifidobacterium and the overestimation of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by poor alpha diversity, a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Our study provides an extended comparative analysis with robust statistical support that could optimize the use of both approaches for gut microbiota research. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes. Both techniques detected an aberrant microbiota in infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment.

The biological variability among Neospora caninum isolates has been widely shown, however, the molecular basis that determines this diversity has not been thoroughly elucidated to date. The latest studies have focused on a limited number of isolates. Therefore, the goal of the present study was to compare the proteome of a larger number of N. caninum isolates with different origins and virulence. Label-free LC-MS/MS was used to investigate the tachyzoite proteomic differences among Nc-Bahia, Nc-Spain4H and Nc-Spain7, representing high virulence isolates and Nc-Ger6, Nc-Spain2H and Nc-Spain1H, representing low virulence isolates. Pairwise comparisons between all isolates and between high virulence and low virulence groups identified a subset of proteins with higher abundance in high virulence isolates. These proteins were involved in energy and redox metabolism, and DNA/RNA processing, which might determine the faster growth rates and parasite survival of the high virulence isolates. Highlighted proteins included a predicted member of the rhoptry kinase family ROP20 specific for N. caninum, Bradyzoite pseudokinase 1 and several dense granule proteins. DNA polymerase, which was more abundant in all high virulence isolates in all comparisons, might also be implicated in virulence. These results reveal insights into possible mechanisms involved in specific phenotypic traits and virulence in N. caninum, and the relevance of these candidate proteins for N. caninum virulence deserves further investigation.

The transition between yeast and hyphal morphologies plays a crucial role in the pathogenicity of Candida albicans. Recent studies have pointed out the great relevance of extracellular vesicles (EVs) secreted by microorganisms in a wide variety of biological processes including interaction with the host. Therefore, the main objective of this work was to compare the EVs secreted by yeast and hyphal forms to shed light on C. albicans-host interaction. EVs were obtained by ultracentrifugation of the culture medium supernatant and analysed by mass spectrometry. They were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS). DLS and TEM analysis showed that yeast EVs were significantly bigger than hyphal EVs, being most of them in the range between 400 to 500nm while hyphal EVs were ranged mostly around 100-200nm. Proteomic analysis showed greater protein diversity in hyphal EVs when compared to yeast EVs (up to 1700 different proteins identified versus 300), although less amount of total protein was obtained. Gene Ontology (GO) analysis showed that yeast EVs were enriched in surface proteins while hyphal EVs, although containing also most of these surface proteins, were also significantly and exclusively enriched in proteins involved in protein metabolism (ribosomal proteins, many aminoacid-pathway enzymes and proteasome) and cellular transport. The differences between YEVs and HEVs also prompted a different immune host response, as tested with macrophage cell cultures and human sera from patients with invasive candidiasis. All these differences point out a possible different biogenesis and roles of EVs secreted by both morphologies.

Metaproteomics is as a promising technique for studying the human gut microbiota, because it can reveal the taxonomic profile and also shed light on the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing and microbial cell disruption methods for metaproteomic analyses of human gut microbiota. An unsupervised principal component analysis showed that different methods produced similar human gut microbial taxonomic profiles. An unsupervised two-way hierarchical clustering analysis identified the microbial taxonomic signatures associated with each method. Proteobacteria and Bacteroidetes identification was favored by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads. Ascomycota identification was enhanced by using large-sized glass beads during sample processing for stool dispersion. Euryarchaeota identification was improved with a combination of small and medium-sized glass beads for cell disruption. Assessments of the relative abundance of Firmicutes, Actinobacteria and Spirochaetes improved when ultrasonication was performed before cell disruption with glass beads. The latter method also increased the overall number of identified proteins. Taxonomic and protein functional analyses of metaproteomic data derived from stool samples from six healthy individuals showed common taxonomic profiles. We also detected certain proteins involved in microbial functions relevant to the host and related mostly to particular taxa, such as B12 biosynthesis and short chain fatty acid production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. Finally, in this metaproteomic study we identified several human proteins, mostly related to the anti-microbial response, which could contribute to determining the beneficial and detrimental relationships between gut microbiota and human cells in particular human diseases.

Extracellular vesicles (EVs) have been described as mediators of microorganism survival and interaction with the host. InCandida albicans, a relevant commensal fungal pathogen, the dimorphic transition is an important virulence trait in candidiasis. We have analyzed EVs secreted by yeast (YEVs) or hyphal cells (HEVs) fromC. albicans, finding interesting differences in both size distribution and protein loading. In general, HEVs were smaller and carried a much more diverse protein cargo than YEVs, including most of the proteins identified in YEVs, which were mainly cell surface proteins. Virulence factors such as phospholipases, aspartic proteases (Saps), adhesins and invasins, and the precursor protein of candidalysin toxin Ece1p were also detected. HEVs were also enriched in proteasomal and ribosomal proteins, and in enzymes from amino acid biosynthetic pathways, all involved in protein metabolism, as well as proteins related to intracellular protein transport and components of the ESCRT pathway related to exosome biogenesis. Both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis. In our conditions, only HEVs could elicit the release of TNFα by activated macrophages. This first analysis ofC. albicansHEVs shows their relevance to pathogenesis and possible new diagnostics or treatments.

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.

To provide new and expanded proteome documentation of the opportunistically pathogen Candida albicans, we have developed new protein extraction and analysis routines to provide a new, extended and enhanced version of the C. albicans PeptideAtlas. Two new datasets, resulting from experiments consisting of exhaustive subcellular fractionations and different growing conditions, plus two additional datasets from previous experiments on the surface and the secreted proteomes, have been incorporated to increase the coverage of the proteome. High resolution precursor mass spectrometry (MS) and ion trap tandem MS spectra were analyzed with three different search engines using a database containing allele-specific sequences. This approach, novel for a large-scale C. albicans proteomics project, was combined with the post-processing and filtering implemented in the Trans Proteomic Pipeline consistently used in the PeptideAtlas project and resulted in 49,372 additional peptides (3-fold increase) and 1630 more proteins (1.6-fold increase) identified in the new C. albicans PeptideAtlas with respect to the previous build. A total of 71,310 peptides and 4174 canonical (minimal non-redundant set) proteins (4115 if one protein per pair of alleles is considered) were identified representing 66% of the 6218 proteins in the predicted proteome. This makes the new PeptideAtlas build the most comprehensive C. albicans proteomics resource available and the only large-scale one with detections of individual alleles., This work has been financially supported in part by project BIO2012-31767Ministerio de Economía y Competitividad, Spain, PROPMT (S2010/BMD-2414) from the Comunidad Autónoma de Madrid, REIPI, Spanish Network for the Research in Infectious Diseases (RD12/0015/0004), and PRB2 projects PT13/0001/0004 and PT13/0001/0008 from the ISCIII. VV held a research contract associated to project BIO2012-31767. These results are lined up with the Spanish Initiative on the Human Proteome Project (B/D-HPP). This work was funded in part by the American Recovery and Reinvestment Act (ARRA) funds through National Institutes of Health from the NHGRI grant RC2HG005805, the NIGMS grants R01GM087221 and 2P50GM076547 to the Center for Systems Biology, the National Institute of Biomedical Imaging and Bioengineering grant U54EB020406, the National Science Foundation MRI grant 0923536, and the EU FP7 ‘ProteomeXchange’ grant 260558.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

18 páginas, 5 figuras, 1 tabla, Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome., Work in our laboratory is supported by the Spanish Ministry of Science and Innovation (BFU2011–27568), Spanish Ministry of Economy and Competitively (BFU2013–43132-P and BFU2016–77975-R AEI/FEDER, UE cofounded by FEDER funds/European Regional Development Fund- a way to build Europe). MRL was supported by the Lundbeck foundation (Junior Group Leader Fellowship). This work was supported by a generous grant from the VILLUM Foundation to the VILLUM Centre for Bioanalytical Sciences at the University of Southern Denmark. SBB is a recipient of ISCIII grant 13FIS037. IDIBELL Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, and is supported by grant PT13/0001/0033.

Pedro J. Navarro et al., The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including 18O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data. © 2014 American Chemical Society., This work was supported by grants BIO2009-07990, BIO2009-11735, BFU2009-08004, SAF 2009-07520, and BIO2012-37926 from the Spanish Ministry of Science and Education, CAM BIO/0194/2006 (Cardiovrep) from the Madrid regional government, and an institutional grant from the Fundación Ramón Areces to the CBMSO. Grants RD06/0014/0030, RD12/0042/0021, RD06/0014/0005, and RD12/0042/0022 from the Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares (RECAVA/RIC, Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Ministry of Health) supported the research of J.V. and J.M.R. P.M.-A. is recipient of a fellowship from the Madrid regional government supported by the European Social Fund

This article belongs to a special issue Special Issue: Standardization and Quality Control in Proteomics: Technical note.-- et al., Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM).

The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC-MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.

The ability to switch from yeast to hyphal growth is essential for virulence in Candida albicans. The cell surface is the initial point of contact between the fungus and the host. In this work, a free-gel proteomic strategy based on tryptic digestion of live yeast and hyphae cells and protein identification using LC-MS/MS methodology was used to identify cell surface proteins. Using this strategy, a total of 943 proteins were identified, of which 438 were in yeast and 928 were in hyphae. Of these proteins, 79 were closely related to the organization and biogenesis of the cell wall, including 28 GPI-anchored proteins, such as Hyr1 and Sod5 which were detected exclusively in hyphae, and Als2 and Sap10which were detected only in yeast. A group of 17 proteins of unknown function were subsequently studied by analysis of the corresponding deletion mutants. We found that four new proteins, Pst3, Tos1, Orf19.3060 and Orf19.5352 are involved in cell wall integrity and in C. albicans' engulfment by macrophages. Moreover, the putative NADH-ubiquinone-related proteins, Ali1, Mci4, Orf19.287 and Orf19.7590, are also involved in osmotic and oxidative resistance, yeast to hypha transition and the ability to damage and invade oral epithelial cells. This article is part of a Special Issue entitled: HUPO 2014.

In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood., Authors would like to acknowledge Joana Tulha for assistance on yeasts overlay photographs, and to Rui Armada for C. albicans ECM SDS-PAGE experiment. The proteomic analysis was carried out at the proteomics facility UCM-PCM, a member of the ProteoRed network. The polysaccharide analysis was performed at the Laboratory of Glycoconjugates Biochemistry and Cellular Biology, UFRJ, Brazil. Fabio Faria-Oliveira was supported by a PhD scholarship SFRH/BD/45368/2008 from FCT (Fundacao para a Ciencia e a Tecnologia). This work was funded by Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by FCT/MEC through Portuguese funds (PIDDAC) - PEst-OE/BIA/UI4050/2014. The authors would also like to acknowledge Hugh S. Johnson for critical reading of the manuscript regarding English usage.

The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5'-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus.

All participating laboratories are members of ProteoRed-ISCIII.-- et al., The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study., This work was supported by: ProteoRed and the Carlos III National Health Institute Agreement, ProteoRed-ISCIII; the agreement between FIMA and the “UTE project CIMA”; grants SAF2011-29312 from Ministerio de Ciencia e Innovación and ISCIII-RETIC RD06/0020 to FJC and EU FP7 grant ProteomeXchange [grant number 260558]. APM and DTM have been funded by Spanish grants from Ministerio de Ciencia e Innovación BIO2010-17527 and the Government of Madrid (P2010/BMD-2305). BBVA Foundation for its support to HUPO initiatives.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2ACdc55 phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2ACdc55 phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2ACdc55 regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2ACdc55 initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2ACdc55 functions affect the regulation of various MEN components, contributing to mitotic exit., Author Summary Cell cycle studies over the years have tried to elucidate the molecular mechanisms behind cell division, one of the most highly regulated of all cell processes, which ensures life in all organisms. Protein phosphorylation emerged as a key regulatory mechanism in the cell cycle. The highly conserved family of cyclin-dependent kinases, the Cdks, are considered the main component of the cell cycle control system. However, it has become clear that opposing phosphatases also play a key role in determining the phosphorylation state of the proteins. Cells enter mitosis when mitotic Cdk activity increases, having its pick of activity during metaphase. To exit mitosis, cells must coordinate chromosome segregation with Cdk inactivation processes involving the activation of protein phosphatases. Here we show that the phosphatase PP2A regulates the mitotic exit network (MEN) by counteracting the phosphorylation of Bfa1 and Mob1. Our findings provide new insights into the mechanism by which PP2A-Cdc55 functions affect the regulation of various MEN components that contribute to mitotic exit. The core signalling elements of the MEN, SIN and Hippo pathways are highly conserved. Therefore, studies of MEN regulation will contribute to our understanding of MEN-related pathways in other organisms.

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.

We have set up a fast and easy methodology to identify cell-surface proteins in live yeasts. A non-gel proteomic approach was based on a short period of trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS. Candida albicans was used as a model organism and proteins involved in cell wall organization, cell rescue, defense, virulence, transport, protein fate and metabolism were identified. This strategy is a powerful tool to study host–pathogen interactions and to look for potential vaccine candidates and drug targets.

The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S–S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC–MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. “Bona fide” cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.

High hydrostatic pressure (HHP) is a new method used to reduce or eliminate microorganisms that are present in food. Proteins are known to be the most important target of high pressure in living organisms. The main goal of this investigation was focused on the changes that occur on the proteins of Bacillus cereus under HHP stress conditions. The two-dimensional differential gel electrophoresis (2D DIGE) technique allows for a simultaneous resolution of thousands of proteins based on fluorescent prelabeling of the samples with spectrally resolvable fluorescent CyDyes. The results of proteomics profiling show an average of 1300 spots being detected. The analysis revealed 75 spot proteins whose abundance is modified after the application of high pressure, of which 66 were decreased after the HHP treatment. Among them, flagellin was the protein that changed the most. The differential expression of some proteins after HHP treatment at 700 MPa may suggest a reduction of virulence and protective response against oxidative stress in flagellated Bacillus., This work has been supported by the National Plan of Spanish I+D+I of the Ministry of Education and Science (MEC) through the project AGL2007-63314/ALI.

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. Methods: S. cerevisiae W303-1A wt strain and gup1Δ mutant were used as previously described to generate biofilmlike mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1Δ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance., This work was funded by the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013. Fábio Faria-Oliveira was supported by a PhD scholarship (SFRH/BD/45368/2008) from FCT (Fundação para a Ciência e a Tecnologia). We thank David Caceres and Montserrat MartinezGomariz from the Unidad de Proteómica, Universidad Complutense de Madrid – Parque Científico de Madrid, Spain for excellent technical assistance in the successful implementation of all proteomics procedures including peptide identification, and Joana Tulha from the CBMA, Universidade do Minho, Portugal, for helping with the SDS-PAGE experiments, and the tedious and laborious ECM extraction procedures. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with the dataset identifier PXD001133. We would like to thank the PRIDE team for all the help and support during the submission process., info:eu-repo/semantics/publishedVersion