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Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with

The definition of the secretome signature of a cancer cell line can be considered a potential tool to investigate tumor aggressiveness and a preclinical exploratory study required to optimize the search of cancer biomarkers. Dealing with a cell-specific secretome limits the contamination by the major components of the human serum and reduces the range of dynamic concentrations among the secreted proteins, thus favouring under-represented tissue-specific species. The aim of the present study is to characterize the secretome of two human colon carcinoma cell lines, CaCo-2 and HCT-GEO, in order to evaluate differences and similarities of two colorectal cancer model systems. In this study, we identified more than 170 protein species, 64 more expressed in the secretome of CaCo-2 cells and 54 more expressed in the secretome of HCT-GEO cells; 58 proteins were shared by the two systems. Among them, more than 50% were deemed to be secretory according to their Gene Ontology annotation and/or to their SignalP or SecretomeP scores. Such a characterization allowed corroborating the potential of a cell culture-based model in order to describe the cell-specific invasive properties and to provide a list of putative cancer biomarkers.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.

The Arctic and Antarctic marine faunas differ by age and isolation. Fishes of the two polar regions have undergone different regional histories that have driven the physiological diversities. Antarctic fish are highly stenothermal, in keeping with stable water temperatures, whereas Arctic fish, being exposed to seasonal temperature variations, exhibit higher physiological plasticity. This study reports the characterization of the oxygen transport system of three Arctic species of the family Gadidae, namely the Arctic cod Arctogadus glacialis, the polar cod Boreogadus saida, and the Atlantic cod Gadus morhua. Unlike Antarctic notothenioids, the blood displays high multiplicity, i.e. it has three hemoglobins, similar to many other acanthomorph teleosts. In the most abundant hemoglobin, oxygen binding is modulated by heterotropic effectors, with marked Bohr and Root effects. Remarkably, in two species (A. glacialis and B. saida), the Hill coefficient is very close to one in the whole pH range, indicating the apparent absence of cooperativity. The amino acid sequences have been used to gain insight into the evolution history of globins of polar fish. The results indicate that Arctic and Antarctic globins have different phylogenies and lead us to suggest that the selective pressure of environment stability allows the phylogenetic signal to be maintained in the Antarctic sequences, whereas environmental variability would tend to disrupt this signal in the Gadidae sequences.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled "Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats". After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243.

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. SIGNIFICANCE: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value.

Paratuberculosis (PTB) or Johne’s disease is a contagious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Ovine PTB is less understood than bovine PTB, especially concerning paucibacillary infection and its evolution into clinical disease. We combined shotgun proteomics, histopathology and immunohistochemistry for the characterization of ileal tissues collected from seven asymptomatic sheep negative to serum ELISA, positive to feces and tissue MAP IS900 and F57 PCR, histologically classified as paucibacillary, actively infected, together with 3 MAP-free controls (K). Following shotgun proteomics with label-free quantitation and differential analysis, 96 proteins were significantly changed in PTB vs K, and were mostly involved in immune defense processes and in the macrophage-MAP interaction. Principal component analysis (PCA) of protein abundances highlighted two PTB sample clusters, PTB1 and PTB2, indicating a dichotomy in their proteomic profiles. This was in line with the PCA of histopathology data and was related to features of type 2 (PTB1) and type 3a (PTB2) lesions, respectively. PTB2 proteomes differed more than PTB1 proteomes from K: 43 proteins changed significantly only in PTB2 and 11 only in PTB1. The differential proteins cathelicidin, haptoglobin, S100A8 and S100A9 were evaluated by immunohistochemistry. K tissues were negative to cathelicidin and haptoglobin and sparsely positive to S100A8 and S100A9. PTB tissues were positive to all four proteins, with significantly more cells in PTB2 than in PTB1. In conclusion, we described several pathways altered in paucibacillary PTB, highlighted some proteomic differences among paucibacillary PTB cases, and identified potential markers for disease understanding, staging, and detection. Electronic supplementary material The online version of this article (10.1186/s13567-018-0612-0) contains supplementary material, which is available to authorized users.

In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis . The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC

""Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W -> F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.""

Background: With the increase in the number of genome sequencing projects, there is a concomitant exponential growth in the number of protein sequences whose function is still unknown. Functional proteomics constitutes an emerging research area in the proteomic field whose approaches are addressed towards two major targets: the elucidation of the biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Methods: The identification of interacting proteins in stable complexes in vivo is essentially achieved by affinity-based procedures. The basic idea is to express the protein of interest with a suitable tag to be used as a bait to fish its specific partners out from a cellular extract. Individual components within the multi-protein complex can then be identified by mass spectrometric methodologies. Results and conclusions: The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism is strongly suggestive of the biological function of the protein. Moreover, the identification of protein partners interacting with a given protein will lead to the description of cellular mechanisms at the molecular level. The next goal will be to generate animal models bearing a tagged form of the bait protein.

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0196-x) contains supplementary material, which is available to authorized users.

Diamond-Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one-fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild-type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild-type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 (http://proteomecentral.proteomexchange.org/dataset/PXD000640).

Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants. © 2013 Cacciotto et al.

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae . A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.

Cancer is a multi-step process, one of the latest events correspond to metastasis formation and dissemination, to date the major cause of deaths. The h-prune-nm23-H1 protein complex and its activation of PDE-cAMP activity have been shown to correlate with breast cancer progression and metastasis formation. Here, we describe the protein complex formation and its involvement in cell migration. By gene expression studies and protein-protein pull-down analyses coupled to mass spectrometry we have identified new genes and pathways along which the h-prune-nm23-H1 complex exerts its function. We review here h-prune binding to the glycogen synthase kinase (GSK-3beta) and identify a new h-prune protein partner, Gelsolin, an ATP severing protein acting in focal adhesions, in a MDA-435 breast cancer cellular model. The results presented here underline the importance of this protein complex leading to new translational studies involved into the inhibition of cell migration, thus enhancing the potential of using this knowledge to direct inhibition of metastases formation in humans.

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1–93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

The term proteome is traditionally associated with the identification of a large number of proteins within complex mixtures originating from a given organelle, cell or even organism. Current proteome investigations are basically focused on two major areas, expression proteomics and functional proteomics. Both approaches rely on the fractionation of protein mixtures essentially by two-dimensional polyacrylamide gel electrophoresis (2D-gel) and the identification of individual protein bands by mass spectrometric techniques (2D-MS). Functional proteomics approaches are basically addressing two main targets, the elucidation of the biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. In the cell many processes are governed not only by the relative abundance of proteins but also by rapid and transient regulation of activity, association and localization of proteins and protein complexes. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular process would then be strongly suggestive of its biological function. The identification of interacting proteins in stable complexes in a cellular system is essentially achieved by affinity-based procedures. Different strategies relying on this simple concept have been developed and a brief overview of the main approaches presently used in functional proteomics studies is described.

Background Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

SummaryOver 40 human diseases are associated with the formation of well-defined proteinaceous fibrillar aggregates. Since the oligomers precursors to the fibrils are increasingly recognized to be the causative agents of such diseases, it is important to elucidate the mechanism of formation of these early species. The acylphosphatase from Sulfolobus solfataricus is an ideal system as it was found to form, under conditions in which it is initially native, two types of prefibrillar aggregates: (1) initial enzymatically active aggregates and (2) oligomers with characteristics reminiscent of amyloid protofibrils, with the latter originating from the structural reorganization of the initial assemblies. By studying a number of protein variants with a variety of biophysical techniques, we have identified the regions of the sequence and the driving forces that promote the first aggregation phase and show that the second phase consists in a cooperative conversion involving the entire globular fold.

[This corrects the article DOI: 10.1371/journal.pone.0276751.]., (Copyright: © 2024 De Forni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)