Mass Spectrometry-based Protein Identification Research Articles

PFKP deubiquitination and stabilization by USP5 activate aerobic glycolysis to promote triple-negative breast cancer progression

BackgroundTriple-negative breast cancer (TNBC) remains the most challenging subtype of breast cancer and lacks definite treatment targets. Aerobic glycolysis is a hallmark of metabolic reprogramming that contributes to cancer progression. PFKP is a rate-limiting enzyme involved in aerobic glycolysis, which is overexpressed in various types of cancers. However, the underlying mechanisms and roles of the posttranslational modification of PFKP in TNBC remain unknown.MethodsTo explore whether PFKP protein has a potential role in the progression of TNBC, protein levels of PFKP in TNBC and normal breast tissues were examined by CPTAC database analysis, immunohistochemistry staining (IHC), and western blotting assay. Further CCK-8 assay, colony formation assay, EDU incorporation assay, and tumor xenograft experiments were used to detect the effect of PFKP on TNBC progression. To clarify the role of the USP5-PFKP pathway in TNBC progression, ubiquitin assay, co-immunoprecipitation (Co-IP), mass spectrometry-based protein identification, western blotting assay, immunofluorescence microscopy, in vitro binding assay, and glycolysis assay were conducted.ResultsHerein, we showed that PFKP protein was highly expressed in TNBC, which was associated with TNBC progression and poor prognosis of patients. In addition, we demonstrated that PFKP depletion significantly inhibited the TNBC progression in vitro and in vivo. Importantly, we identified that PFKP was a bona fide target of deubiquitinase USP5, and the USP5-mediated deubiquitination and stabilization of PFKP were essential for cancer cell aerobic glycolysis and TNBC progression. Moreover, we found a strong positive correlation between the expression of USP5 and PFKP in TNBC samples. Notably, the high expression of USP5 and PFKP was significantly correlated with poor clinical outcomes.ConclusionsOur study established the USP5-PFKP axis as an important regulatory mechanism of TNBC progression and provided a rationale for future therapeutic interventions in the treatment of TNBC.

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.

Off-the-shelf proximity biotinylation using ProtA-TurboID.

Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or clustered regularly interspaced short palindromic repeats (CRISPR)-based tagging, thus prohibiting the use of such assays in cell types that are difficult to transfect or transduce. We recently developed an 'off-the-shelf' proximity biotinylation method that makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait-specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. In principle, any scientist can perform this protocol within 3 days, although generating the proteomics data requires access to a high-end liquid chromatography-mass spectrometry setup. Data analysis and data visualization are relatively straightforward and can be performed using any type of software that converts raw mass spectrometry spectra files into identified and quantified proteins. The protocol has been optimized for nuclear targets but may also be adapted to other subcellular regions of interest.

Proteomics-based identification of cancer-associated proteins in chronic lymphocytic leukaemia

Background: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. Results: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) ≤ 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing ≤ 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p Conclusions: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

The interactome of Singapore grouper iridovirus protein ICP18 as revealed by proximity-dependent BioID approach

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that is a major threat to grouper aquaculture. The pathogenesis of SGIV is not well understood so far. Previous studies have revealed that ICP18, an immediate early protein encoded by SGIV ORF086R gene, promotes viral replication by regulating cell proliferation and virus assembly. In the present study, the potential functions of ICP18 were further explored by probing into its interactors using a proximity-dependent BioID method. Since our in-house grouper embryonic cells (a natural host cell of SGIV) could not be efficiently transfected with the plasmid DNA, and the grouper genome data for mass spectrometry-based protein identification is not currently available, we chosen a non-permissive cell (HEK293 T) as a substitute for this study. A total of 112 cellular proteins that potentially bind to ICP18 were identified by mass spectrometry analysis. Homology analysis showed that among these identified proteins, 110 candidate ICP18-interactors had homologous proteins in zebrafish (a host of SGIV), and shared high sequence identity. Further analysis revealed that the identified ICP18-interacting proteins modulate various cellular processes such as cell cycle and cell adhesion. In addition, the interaction between ICP18 and its candidate interactor, i.e., cyclin-dependent kinase1 (CDK1), was confirmed using Co-immunoprecipitation (Co-IP) and Pull-down assays. Collectively, our present data provides additional insight into the biological functions of ICP18 during viral infection, which could help in further unraveling the pathogenesis of SGIV.

Pioglitazone corrects dysregulation of skeletal muscle mitochondrial proteins involved in ATP synthesis in type 2 diabetes

ContextIn this study, we aimed to identify the determinants of mitochondrial dysfunction in skeletal muscle (SKLM) of subjects with type 2 diabetes (T2DM), and to evaluate the effect of pioglitazone (PIO) on SKLM mitochondrial proteome. MethodsTwo different groups of adults were studied. Group I consisted of 8 individuals with normal glucose tolerance (NGT) and 8 with T2DM, subjected to SKLM mitochondrial proteome analysis by 2D-gel electrophoresis followed by mass spectrometry-based protein identification. Group II included 24 individuals with NGT and 24 with T2DM, whose SKLM biopsies were subjected to immunoblot analysis. Of the 24 subjects with T2DM, 20 were randomized to receive placebo or PIO (15 mg daily) for 6 months. After 6 months of treatment, SKLM biopsy was repeated. ResultsMitochondrial proteomic analysis on Group I revealed that several mitochondrial proteins involved in oxidative metabolism were differentially expressed between T2DM and NGT groups, with a downregulation of ATP synthase alpha chain (ATP5A), electron transfer flavoprotein alpha-subunit (ETFA), cytochrome c oxidase subunit VIb isoform 1 (CX6B1), pyruvate dehydrogenase protein X component (ODPX), dihydrolipoamide dehydrogenase (DLDH), dihydrolipoamide-S-succinyltransferase (DLST), and mitofilin, and an up-regulation of hydroxyacyl-CoA-dehydrogenase (HCDH), 3,2-trans-enoyl-CoA-isomerase (D3D2) and delta3,5-delta2,4-dienoyl-CoA-isomerase (ECH1) in T2DM as compared to NGT subjects. By immunoblot analysis on SKLM lysates obtained from Group II we confirmed that, in comparison to NGT subjects, those with T2DM exhibited lower protein levels of ATP5A (−30%, P = 0.006), ETFA (−50%, P = 0.02), CX6B1 (−30%, P = 0.03), key factors for ATP biosynthesis, and of the structural protein mitofilin (−30%, P = 0.01). T2DM was associated with a reduced abundance of the enzymes involved in the Krebs cycle DLST and ODPX (−20%, P ≤ 0.05) and increased levels of HCDH and ECH1, enzymes implicated in the fatty acid catabolism (+30%, P ≤ 0.05).In subjects with type 2 diabetes treated with PIO for 6 months we found a restored SKLM protein abundance of ATP5A, ETFA, CX6B1, and mitofilin. Moreover, protein levels of HCDH and ECH1 were reduced by −10% and − 15% respectively (P ≤ 0.05 for both) after PIO treatment. ConclusionType 2 diabetes is associated with reduced levels of mitochondrial proteins involved in oxidative phosphorylation and an increased abundance of enzymes implicated in fatty acid catabolism in SKLM. PIO treatment is able to improve SKLM mitochondrial proteomic profile in subjects with T2DM.

Bioinformatics Methods for Mass Spectrometry-Based Proteomics Data Analysis.

Recent advances in mass spectrometry (MS)-based proteomics have enabled tremendous progress in the understanding of cellular mechanisms, disease progression, and the relationship between genotype and phenotype. Though many popular bioinformatics methods in proteomics are derived from other omics studies, novel analysis strategies are required to deal with the unique characteristics of proteomics data. In this review, we discuss the current developments in the bioinformatics methods used in proteomics and how they facilitate the mechanistic understanding of biological processes. We first introduce bioinformatics software and tools designed for mass spectrometry-based protein identification and quantification, and then we review the different statistical and machine learning methods that have been developed to perform comprehensive analysis in proteomics studies. We conclude with a discussion of how quantitative protein data can be used to reconstruct protein interactions and signaling networks.

Mass spectrometry-based protein identification in proteomics-a review.

Statistically, accurate protein identification is a fundamental cornerstone of proteomics and underpins the understanding and application of this technology across all elements of medicine and biology. Proteomics, as a branch of biochemistry, has in recent years played a pivotal role in extending and developing the science of accurately identifying the biology and interactions of groups of proteins or proteomes. Proteomics has primarily used mass spectrometry (MS)-based techniques for identifying proteins, although other techniques including affinity-based identifications still play significant roles. Here, we outline the basics of MS to understand how data are generated and parameters used to inform computational tools used in protein identification. We then outline a comprehensive analysis of the bioinformatics and computational methodologies used in protein identification in proteomics including discussing the most current communally acceptable metrics to validate any identification.

Antioxidant activity and identification of food proteins by digestive enzyme supplementation and fermentation with Lactobacillus kefiri

Casein, gluten, and soy protein are widely used in food processing for structure, texture, and flavour improvement. These large proteins might be hydrolysed to shorter peptides or amino acids, which provide antioxidant activities through enzymatic and fermentative food processes. Casein, gluten, and soy protein were digested with an enzyme supplement product containing dipeptidyl peptidase IV (DPPIV) and protease in this study. Then, each protein was hydrolysed by Lactobacillus kefiri strain. 2,2, diphenyl 1-picryl hydrazyl (DPPH) radical scavenging activity and reducing power (RP) were measured for undigested and digested samples. According to our results, all proteins were hydrolysed. Soy protein demonstrated the highest IC<sub>50</sub> value of DPPH for undigested (2.64 mg/ml) and digested samples (1.56 mg/ml) as well as the highest RP value (0.171 for undigested and 0.234 for digested at 700 nm). On the other hand, casein provided the weakest DPPH radical scavenging activity (1.58 ± 0.041% for undigested and 21.86 ± 0.012% for digested samples). A strong correlation was found between cell growth and antioxidant activity of casein during the microbial fermentation. In addition, the changes in protein expression levels by microbial fermentation were analysed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry-based protein identification studies revealed that EF-G, DNA-K, and DNA-J were mainly overexpressed indicating L. kefiri adapts itself by changing the overall proteome.

Identification of Chitinase-3-Like Protein 1 as a Novel Neutrophil Antigenic Target in Crohn’s Disease

Background and AimsThere is an increasing incidence of inflammatory bowel disease [IBD]. Autoimmune responses are involved in the pathophysiology of IBD, but their underlying pathways and target antigens have not yet been fully elucidated.MethodsAutoantigenic targets in IBD were identified after separation of whole cell proteins isolated from neutrophils using two-dimensional electrophoresis and matrix assisted laser desorption ionization – time of flight mass spectrometry-based protein identification of the spots that displayed Western blotting signals with anti-neutrophil cytoplasmic antibody-positive sera. The prevalence of IgG, IgA and secretory IgA [sIgA] to chitinase 3-like protein 1 [CHI3L1] was analysed by enzyme-linked immunosorbent assays using recombinant CHI3L1 in 110 patients with Crohn’s disease [CD], 95 with ulcerative colitis [UC], 126 with coeliac disease [CeD] and 86 healthy controls [HCs].ResultsThe 18-glycosylhydrolase family member CHI3L1 was identified as a neutrophil autoantigenic target. CD patients displayed significantly higher levels of IgG to CHI3L1 than patients with UC and CeD (p < 0.0001, respectively). IgA and sIgA to CHI3L1 was significantly higher in CD than in UC, CeD and HCs [p < 0.0001, respectively]. IgA and sIgA to CHI3L1 demonstrated the highest prevalence in CD [25.5%, 28/110; and 41.8%%, 46/110] compared to HCs [2.3%, 2/86; and 4.7%%, 4/86; p = 0.0015 and p < 0.0001] and are associated with a more complicated progression of CD.ConclusionCHI3L1 is a novel neutrophil autoantigenic target in CD. IgA and sIgA to CHI3L1 may serve as novel markers for CD and may facilitate the serological diagnosis of IBD.

Metal complexation by histidine-rich peptides confers protective roles against cadmium stress in Escherichia coli as revealed by proteomics analysis.

The underlying mechanism and cellular responses of bacteria against toxic cadmium ions is still not fully understood. Herein, Escherichia coli TG1 expressing hexahistidine-green fluorescent protein (His6GFP) and cells expressing polyhistidine-fused to the outer membrane protein A (His-OmpA) were applied as models to investigate roles of cytoplasmic metal complexation and metal chelation at the surface membrane, respectively, upon exposure to cadmium stress. Two-dimensional gel electrophoresis (2-DE) and two-dimensional difference in gel electrophoresis (2D-DIGE) in conjunction with mass spectrometry-based protein identification had successfully revealed the low level expression of antioxidative enzymes and stress-responsive proteins such as manganese-superoxide dismutase (MnSOD; +1.65 fold), alkyl hydroperoxide reductase subunit C (AhpC; +1.03 fold) and DNA starvation/stationary phase protection protein (Dps; −1.02 fold) in cells expressing His6GFP in the presence of 0.2 mM cadmium ions. By contrarily, cadmium exposure led to the up-regulation of MnSOD of up to +7.20 and +3.08 fold in TG1-carrying pUC19 control plasmid and TG1 expressing native GFP, respectively, for defensive purposes against Cd-induced oxidative cell damage. Our findings strongly support the idea that complex formation between cadmium ions and His6GFP could prevent reactive oxygen species (ROS) caused by interaction between Cd2+ and electron transport chain. This coincided with the evidence that cells expressing His6GFP could maintain their growth pattern in a similar fashion as that of the control cells even in the presence of harmful cadmium. Interestingly, overexpression of either OmpA or His-OmpA in E. coli cells has also been proven to confer protection against cadmium toxicity as comparable to that observed in cells expressing His6GFP. Blockage of metal uptake as a consequence of anchored polyhistidine residues on surface membrane limited certain amount of cadmium ions in which some portion could pass through and exert their toxic effects to cells as observed by the increased expression of MnSOD of up to +9.91 and +3.31 fold in case of TG1 expressing only OmpA and His-OmpA, respectively. Plausible mechanisms of cellular responses and protein mapping in the presence of cadmium ions were discussed. Taken together, we propose that the intracellular complexation of cadmium ions by metal-binding regions provides more efficiency to cope with cadmium stress than the blockage of metal uptake at the surface membrane. Such findings provide insights into the molecular mechanism and cellular adaptation against cadmium toxicity in bacteria.

Analysis of DNAs associated with coconut foliar decay disease implicates a unique single-stranded DNA virus representing a new taxon

The unique ecology, pathology and undefined taxonomy of coconut foliar decay virus (CFDV), found associated with coconut foliar decay disease (CFD) in 1986, prompted analyses of old virus samples by modern methods. Rolling circle amplification and deep sequencing applied to nucleic acid extracts from virion preparations and CFD-affected palms identified twelve distinct circular DNAs, eleven of which had a size of about 1.3 kb and one of 641 nt. Mass spectrometry-based protein identification proved that a 24 kDa protein encoded by two 1.3 kb DNAs is the virus capsid protein with highest sequence similarity to that of grabloviruses (family Geminiviridae), even though CFDV particles are not geminate. The nine other 1.3 kb DNAs represent alphasatellites coding for replication initiator proteins that differ clearly from those encoded by nanovirid DNA-R. The 641 nt DNA-gamma is unique and may encode a movement protein. Three DNAs, alphasatellite CFDAR, capsid protein encoding CFDV DNA-S.1 and DNA-gamma share sequence motifs near their replication origins and were consistently present in all samples analysed. These DNAs appear to be integral components of a possibly tripartite CFDV genome, different from those of any Geminiviridae or Nanoviridae family member, implicating CFDV as representative of a new genus and family.

Comparative proteome analysis of skeletal muscle between Merino and Tsigai lambs

Proteomic methods allow detecting breed-specific differences at protein level. The objective of the present study was to compare muscle proteome profiles of Hungarian Merino and Tsigai sheep breeds by two dimensional gel electrophoresis and mass spectrometry based protein identification. Approximately 327 protein spots were detected in musculus longissimus dorsi and 14 protein spots, identified as 12 proteins, showed significant differences in their intensity (P<0.05) between breeds. Seven and five proteins had higher expression in Merino and Tsigai skeletal muscle, respectively. Identified proteins were classified as structural, carbohydrate metabolism-related and miscellaneous ones. Six genes, which represent the three protein groups by function, were analysed by quantitative real-time PCR. All of the identified structural proteins have shown higher intensity in Merino, while the expression of identified glycolytic enzymes and myoglobin were higher in Tsigai lambs. One of the structural and one of the carbohydrate metabolism-related protein showed differences in expression at mRNA level by quantitative real-time PCR, as well. To the best of our knowledge, it was the first differential proteome analysis of sheep muscle and these results contribute to a better understanding of molecular differences between breeds.

The human VGF-derived bioactive peptide TLQP-21 binds heat shock 71 kDa protein 8 (HSPA8)on the surface of SH-SY5Y cells.

VGF (non-acronymic)is a secreted chromogranin/secretogranin that gives rise to a number of bioactive peptides by a complex proteolysis mechanism. VGF-derived peptides exert an extensive array of biological effects in energy metabolism, mood regulation, pain, gastric secretion function, reproduction and, perhaps, cancer. It is therefore surprising that very little is known about receptors and binding partners of VGF-derived peptides and their downstream molecular mechanisms of action. Here, using affinity chromatography and mass spectrometry-based protein identification, we have identified the heat shock cognate 71 kDa protein A8 (HSPA8)as a binding partner of human TLQP-21 on the surface of human neuroblastomaSH-SY5Y cells. Binding of TLQP-21 to membrane associated HSPA8 in live SH-SY5Y cells was further supported by cross-linking to live cells. Interaction between HSPA8 and TLQP-21 was confirmed in vitro by label-free Dynamic Mass Redistribution (DMR) studies. Furthermore, molecular modeling studies show that TLQP-21 can be docked into the HSPA8 peptide binding pocket. Identification of HSPA8 as a cell surface binding partner of TLQP-21 opens new avenues to explore the molecular mechanisms of its physiological actions, and of pharmacological modulation thereof.

Microwave & magnetic proteomics of macrophages from patients with HIV-associated cognitive impairment.

ObjectiveHIV-infected monocytes can infiltrate the blood brain barrier as differentiated macrophages to the central nervous system, becoming the primary source of viral and cellular neurotoxins. The final outcome is HIV-associated cognitive impairment (HACI), which remain prevalent today, possibly due to the longer life-span of the patients treated with combined anti-retroviral therapy. Our main goal was to characterize the proteome of monocyte-derived macrophages (MDM) from HACI patients, and its association with their cognitive status, to find novel targets for therapy.MethodsMDM were isolated from the peripheral blood of 14 HIV-seropositive women characterized for neurocognitive function, including: four normal cognition (NC), five asymptomatic (A), and five with cognitive impaired (CI). Proteins from macrophage lysates were isobaric-labeled with the microwave and magnetic (M2) sample preparation method followed by liquid chromatography-tandem mass spectrometry-based protein identification and quantification. Differences in protein abundance across groups classified by HACI status were determined using analysis of variance.ResultsA total of 2,519 proteins were identified with 2 or more peptides and 28 proteins were quantified as differentially expressed. Statistical analysis revealed increased abundance of 17 proteins in patients with HACI (p<0.05), including several enzymes associated to the glucose metabolism. Western blot confirmed increased expression of 6-Phosphogluconate dehydrogenase and L-Plastin in A and CI patients over NC and HIV seronegatives.ConclusionsThis is the first quantitative proteomics study exploring the changes in protein abundance of macrophages isolated from patients with HACI. Further studies are warranted to determine if these proteins may be target candidates for therapy development against HACI.

The proteomic profile of a mouse model of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) develops as a complication of retinal detachment surgery and represents a devastating condition leading to serious vision loss. A good animal model that permits extensive functional studies and drug testing is crucial in finding better therapeutic modalities for PVR. A previously established mouse model, using dispase injection, was analyzed from the proteomic point of view, examining global protein profile changes by 2D electrophoresis, image analysis and HPLC–tandem mass spectrometry‐based protein identification. The easy applicability of the mouse model was used to study the role of transglutaminase 2 (TG2) in PVR formation by proteomic examination of dispase‐induced TG2 knockout vitreous samples. Our data demonstrate that, despite the altered appearance of crystallin proteins, the lack of TG2 did not prevent the development of PVR.

Analysis of Pseudomonas aeruginosa PAO1 Biofilm Protein Profile After Exposure to n-Butanolic Cyclamen coum Extract Alone and in Combination with Ciprofloxacin.

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

Stable isotope dimethyl labelling for quantitative proteomics and beyond.

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized.This article is part of the themed issue 'Quantitative mass spectrometry'.

Tralopyril bioconcentration and effects on the gill proteome of the Mediterranean mussel Mytilus galloprovincialis

Antifouling (AF) systems are used worldwide as one of the most cost-effective ways of protecting submerged structures against heavy biofouling. The emergence of environmentally friendly AF biocides requires knowledge on their environmental fate and toxicity. In this study we measured the bioconcentration of the emerging AF biocide tralopyril (TP) in the Mediterranean mussel Mytilus galloprovincialis and investigated the effects of TP on the mussel gill proteome following acute (2days) and chronic (30days) exposure, as well as after a 10-day depuration period. The experiments were carried out with 1μg/L TP; blank and solvent (5×10−5% DMSO) controls were also included. Proteomics analysis was performed by mass spectrometry-based multidimensional protein identification technology (MudPIT). Differentially expressed proteins were identified using a label-free approach based on spectral counts and G-test. Our results show that TP is rapidly accumulated by mussels at concentrations up to 362ng/g dw (whole tissues), reaching steady-state condition within 13days. Ten days of depuration resulted in 80% elimination of accumulated TP from the organism, suggesting that a complete elimination could be reached with longer depuration times. In total, 46 proteins were found to be regulated in the different exposure scenarios. Interestingly, not only TP but also DMSO alone significantly modulated the protein expression in mussel gills following acute and chronic exposure. Both compounds regulated proteins involved in bioenergetics, immune system, active efflux and oxidative stress, often in the opposite way. Alterations of several proteins, notably several cytoskeletal ones, were still observed after the depuration period. These may reflect either the continuing chemical effect due to incomplete elimination or an onset of recovery processes in the mussel gills. Our study shows that exposure of adult mussels to sublethal TP concentration results in the bioconcentration of this biocide in the tissues and modulates the expression of several proteins that may intervene in important metabolic pathways.

A Deep Insight into the Sialome of Rhodnius neglectus, a Vector of Chagas Disease.

BackgroundTriatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses.Methods/Principal FindingsNext generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant.Conclusions/SignificanceThe data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.