Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labelling approach. We herein describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labelling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis cohort. Two proteins, GAP43 and SERPINA3, were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in late stage secondary progressive multiple sclerosis compared to early stages of multiple sclerosis and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all multiple sclerosis patients compared to controls. The developed assay procedure now offers new possibilities for broad scale protein profiling of CSF within neurological disorders.

Hepatic ischemia-reperfusion (IR) injury is a common clinical problem and reactive oxygen species (ROS) may be a contributing factor on IR injury. The current study evaluates the potential protective effect of saffron ethanol extract (SEE) in a rat model upon hepatic IR injury. Caspases 3 and TUNEL's results showed increased cell death in the IR samples; reversely, minor apoptosis was detected in the SEE/IR group. Pretreatment with SEE significantly restored the content of antioxidant enzymes (SOD1 and catalase) and remarkably inhibited the intracellular ROS concentration in terms of reducing p47phox translocation. Proteome tools revealed that 20 proteins were significantly modulated in protein intensity between IR and SEE/IR groups. Particularly, SEE administration could attenuate the carbonylation level of several chaperon proteins. Network analysis suggested that saffron extract could alleviate IR-induced endoplasmic reticulum (ER) stress and protein ubiquitination which finally lead to cell apoptosis. Taken together, SEE could reduce hepatic IR injury through modulating protein oxidation and our results might help to develop novel therapeutic strategies against ROS-caused diseases.

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, MMP-9 and LCN2, which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.

About three decades ago, profilin 1 was described as a 15 kDa small protein. It was later shown that profilin 1 is a tumor suppressor in human carcinomas. Recent proteome based data additionally demonstrated that the levels of profilin 1 expression could help to predict malignant tumor aggressiveness, response to anti-cancer therapy and risk of recurrence development.

The presence of given antigens in environmental samples (e.g. biodegradative enzymes) report the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system, consisting of a complete platform for isolation, amplification and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e. g. catabolic enzymes) for generating a phage display library of variable V_HH domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western-panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, V_HHs are fused to the C-domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3-dioxygenase (BphC), a key enzyme for biodegradation of polychlorinated biphenyls (PCBs). The thereby generated anti-BphC V_HHs revealed the presence of this enzyme in the metaproteome an oil refinery waste treatment plant.

The existence of a gamma-glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma-glutamyl transferase (GGT)-assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular-weight range compared to WT. The combined iTRAQ and LC-MS/MS based quantitative proteomics approach identified 70 proteins (out of 1,013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH-associated genes showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism and photosynthesis, with strong implications for plant's adaptation to environment.

Salinity is one of the major abiotic stresses affecting plant productivity but surprisingly, a thorough understanding of the salt-responsive networks responsible for sustaining growth and maintaining crop yield remains a significant challenge. Rice suspension culture cells (SCCs), a single cell type, were evaluated as a model system as they provide a ready source of a homogenous cell type and avoid the complications of multicellular tissue types in planta. A combination of growth performance, and transcriptional analyses using known salt-induced genes was performed on control and 100 mM NaCl cultured cells to validate the biological system. Protein profiling was conducted using both DIGE- and iTRAQ-based proteomics approaches. In total, 106 proteins were identified in DIGE experiments and 521 proteins in iTRAQ experiments with 58 proteins common to both approaches. Metabolomic analysis provided insights into both developmental changes and salt-induced changes of rice SCCs at the metabolite level; 134 known metabolites were identified, including 30 amines and amides, 40 organic acids, 40 sugars, sugar acids and sugar alcohols, 21 fatty acids and sterols and 3 miscellaneous compounds. Our results from proteomic and metabolomic studies indicate that the salt-responsive networks of rice SCCs are extremely complex and share some similarities with thee cellular responses observed in planta. For instance, carbohydrate and energy metabolism pathways, redox signaling pathways, auxin/IAA pathways and biosynthesis pathways for osmoprotectants are all salt responsive in SCCs enabling cells to maintain cellular function under stress condition. These data are discussed in the context of our understanding of in planta salt-responses.

Proteogenomics sensu stricto refers to the use of proteomic data to refine the annotation of genomes from model organisms. Because of the limitations of automatic annotation pipelines, a relatively high number of errors occur during the structural annotation of genes coding for proteins. Whether putative orphan sequences or short genes encoding low molecular weight proteins really exist is still frequently a mystery. Whether start codons are well defined is also an open debate. These problems are exacerbated for genomes of microorganisms belonging to poorly documented genera, as related sequences are not always available for homology-guided annotation. The functional annotation of a significant proportion of genes is also another well-known issue when annotating environmental microorganisms. High-throughput shotgun proteomics has recently greatly evolved, allowing the exploration of the proteome from any microorganism at an unprecedented depth. The structural and functional annotation process may be usefully complemented with experimental data. Indeed, proteogenomic mapping has been successfully performed for a wide variety of organisms. Specific approaches devoted to systematically establishing the N-termini of a large set of proteins are being developed. N-terminomics is giving rise to datasets of experimentally proven translational start codons, as well as validated peptide signals for secreted proteins. By extension, combining genomic and proteomic data is becoming routine in many research projects. The proteomic analysis of organisms with unfinished genome sequences, the so-called composite proteomics, and the search for microbial biomarkers by bottom-up and top-down combined approaches are some examples of proteogenomic-flavored studies. They illustrate the advent of a new era of environmental microbiology where proteomics and genomics are intimately integrated to answer key biological questions.

Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to non-depletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under non-limiting conditions. Integrated proteomic and metabolomic analysis identified 1,747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase.

Metaproteomics of microbial communities bears the promise to add functional information to the blueprint of genes derived from metagenomics. Right from its beginning the achievements and developments in metaproteomics were closely interlinked with metagenomics. In addition, the evaluation, visualisation and interpretation of metaproteome data demanded for the developments in bioinformatics. This review will give an overview about recent strategies to use genomic data either from public databases or organismal-specific genomes/metagenomes to increase the number of identified proteins obtained by mass spectrometric measurements. We will review different published metaproteogenomic approaches in respect to the used mass spectrometry pipeline and to the used protein identification workflow. Furthermore different approaches of data visualisation and strategies for phylogenetic interpretation of metaproteome data are discussed as well as approaches for functional mapping of the results to the investigated biological systems. This information will in the end allow to comprehensively analysing interactions and interdependencies within microbial communities.

Pseudomonas putida is a ubiquitous, metabolically very versatile, Gram-negative bacterium adapted to habitats as diverse as soil, water and the rhizosphere. Most strains are non-pathogenic, many are used as experimental models, and many others have biotechnological applications in the areas of agriculture, bioremediation, biocatalysis and the production of bioplastics. This review summarises the contribution of proteomic technologies to our understanding of how P. putida responds to different carbon sources, how it adapts to living at sub-optimal temperatures or attached to surfaces, and how it responds to the presence of toxic compounds such as aromatic molecules and heavy metals. The examples described illustrate the value of proteomics in furthering our knowledge of the physiology and behaviour of bacteria, knowledge that is important for understanding how they behave in their natural habitats and for optimising their behaviour in biotechnological applications.

Contemporary protein microarrays like the ProtoArray® are used for autoimmune antibody screening studies to discover biomarker panels. For ProtoArray data analysis the software Prospector and a default workflow are suggested by the manufacturer. While analyzing a large data set of a discovery study for diagnostic biomarkers of the Parkinson's Disease (“ParkCHIP”) we have revealed the need for distinct improvements of the suggested workflow concerning raw data acquisition, normalization and pre-selection method availability, batch effects, feature selection, and feature validation. In this work appropriate improvements of the default workflow are proposed and it is shown that completely automatic data acquisition as a batch, a re-implementation of Prospector's pre-selection method, multivariate or hybrid feature selection, and validation of the selected protein panel using an independent test set define in combination an improved workflow for large studies.

A key global challenge for plant biotechnology is addressing food security, whereby provision must be made to feed 9 billion people with nutritional feedstuffs by 2050. To achieve this step-change in Agricultural production new crop varieties are required that are tolerant to environmental stresses imposed by climate change, have better yields, are more nutritious and require less resource input. Genetic modification (GM) and Marker Assisted Screening (MAS) will need to be fully utilised to deliver these new crop varieties. To evaluate these varieties both in terms of environmental and food safety and the ration design of traits a systems level characterisation is necessary. To link the transcriptome to the metabolome, quantitative proteomics is required. Routine quantitative proteomics is an important challenge. Gel-based densitometry and MS analysis after stable isotope labeling have been employed. In the present article we describe the application of a label-free approach that can be used in combination with SDS-PAGE and reverse-phase chromatography to evaluate the changes in the proteome of new crop varieties. The workflow has been optimised for protein coverage, accuracy, and robustness, then its application demonstrated using a GM tomato variety engineered to deliver nutrient dense fruit.

Trypsin shaving is a targeted proteomic method for identifying cell-surface exposed proteins on bacterial cells. For the identification of redox-active cell-surface proteins, trypsin shaving datasets can be matched with surface-enhanced Raman spectra of intact cells to identify the cofactors associated with the cell-surface proteins. Together, these approaches could help resolve questions about the presence of cell-surface electron transport components in environmental microorganisms, especially microbes that oxidize and reduce metals and metalloids as electron donors and acceptors.

Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either ¹³C-naphthalene or ¹³C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of an PAH contaminated aquifer. Metaproteomic analysis and protein-SIP revealed Burkholderiales, Actinomycetales and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative ¹³C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-SIP may be a significant tool for in situ identification of metabolic key players as well as degradation pathways.

Phaeobacter gallaeciensis DSM 17395, a member of the Roseobacter clade, was studied for its adaptive strategies to complex and excess nutrient supply, here mimicked by cultivation with Marine Broth (MB). During growth in process-controlled fermenters, P. gallaeciensis grew faster (3.6-fold higher μ_max) and reached higher optical densities (2.2-fold) with MB medium, as compared to the reference condition of glucose-containing mineral medium. Apparently, in the presence of MB medium, metabolism was tuned to maximize growth rate at the expense of efficiency. Comprehensive proteomic analysis of cells harvested at ½ OD_max identified 1,783 (2D DIGE, membrane and extracellular protein-enriched fractions, shotgun) different proteins (46% coverage), 315 (based on 2D DIGE) of which displayed differential abundance profiles. Moreover, 145 different metabolites (intra- and extracellular combined) were identified, almost all of which (138) showed abundance changes. During growth with MB medium, P. gallaeciensis specifically formed the various proteins required for utilization of phospholipids and several amino acids, as well as for gluconeogenesis. Metabolic tuning on amino acid utilization is also reflected by massive discharge of urea to dispose the cell of excess ammonia. Apparently, P. gallaeciensis modulated its metabolism to simultaneously utilize diverse substrates from the complex nutrient supply.

The occurrence of high ozone concentrations during drought episodes is common considering that they are partially caused by the same meteorological phenomena. It was suggested that mild drought could protect plants against ozone-induced damage by causing the closure of stomata and preventing the entry of ozone into the leaves. The present experiment attempts to create an overview of the changes in cellular processes in response to ozone, mild drought and a combined treatment based on the use of 2D-DiGE to compare the involved proteins, and a number of supporting analyses. Morphological symptoms were worst in the combined treatment, indicating a severe stress, but fewer proteins were differentially abundant in the combined treatment than for ozone alone. Stomatal conductance was slightly lowered in the combined treatment. Shifts in carbon metabolism indicated that the metabolism changed to accommodate for protective measures and changes in the abundance of proteins involved in redox protection indicated the presence of an oxidative stress. This study allowed to identify a set of proteins that changed similarly during ozone and drought stress, indicative of crosstalk in the molecular response of plants exposed to these stresses. The abundance of other key proteins changed only when the plants are exposed to specific conditions. Together this indicates the coexistence of generalized and specialized responses to the different conditions.

15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) is an endogenous anti-inflammatory lipid derived from PGD₂. One potential mechanism for its activity is the covalent modification of cellular proteins, via a reactive α,β-unsaturated carbonyl group in its cyclopentenone ring, which in turn alters protein function. In order to identify the candidate target proteins covalently modified by 15d-PGJ2 in human aortic endothelial cell (EC), EC were treated with biotinylated-15d-PGJ2, the modified proteins extracted by Neutravidin affinity-purification and the proteins identified by LTQ Orbitrap mass spectrometer. Classification of the 358 identified proteins was performed using PANTHER classification system (www.pantherdb.org), showing that the proteins mapped to metabolic process, cellular process and transport activity. This protein data set highlights the potential for 15d-PGJ2 to covalently modify cellular proteins and provides a source of data that will aid further studies on the mechanism of action of this endogenous regulator of inflammation.

In the present study, we screened proteomic and cytokine biomarkers between patients with adenomatous polyps and colorectal cancer (CRC) in order to improve our understanding of the molecular mechanisms behind turmorigenesis and tumor progression in CRC. To this end, we performed comparative proteomic analysis of plasma proteins using a combination of 2-DE and mass spectrometry as well as profiled differentially regulated cytokines and chemokines by multiplex bead analysis. Proteomic analysis identified 11 up-regulated and 13 down-regulated plasma proteins showing significantly different regulation patterns with diagnostic potential for predicting progression from adenoma to carcinoma. Some of these proteins have not previously been previously implicated in CRC, including up-regulated leucine-rich α-2-glycoprotein, hemoglobin subunit β, Ig α-2 chain C region, and complement factor B as well as down-regulated afamin, zinc-α-2-glycoprotein, vitronectin, and α1-antichymotrypsin. In addition, plasma levels of three cytokines/chemokines, including interleukin-8, interferon gamma-induced protein 10, and tumor necrosis factor α, were remarkably elevated in patients with CRC compared to those with adenomatous polyps. Although further clinical validation is required, these proteins and cytokines can be established as novel biomarkers for CRC and/or its progression from colon adenoma.

The new energetic requirements of the growing world population together with the actual ecological trend of phytoremediation has made challenging the cultivation of energetic crops on non-agricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterisation and biochemical analyses were combined to emphasise the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni and Zn concentrations. Leaves and roots of sunflowers reaching the stage ‘2-extended leaves’ and exposed to different trace metal concentrations were harvested and analysed by 2D-DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reaction and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as remediative tool of trace element-polluted soils is limited.

The current treatment for glioblastoma (GBM) includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ induced changes in the proteome of the glioma derived cell line (U251) by 2D-DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. Mass spectrometry identified four up-regulated spots: aspartyl tRNA synthetase (DARS) glutathione synthetase (GSS), interleukin-1 receptor associated kinase- 4 (IRAK4), and breast carcinoma amplified sequence-1 (BCAS1) and one down-regulated spot: optineurin (OPTN). TMZ induced regulation of these five genes was validated by RT-qPCR and western blot analysis. RNAi mediated knockdown of IRAK4, an important mediator of Toll-like receptors (TLR) signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus we have identified several TMZ modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit TLR signaling and NFkB pathway

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day seven after damage and proteins were separated using 2-DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes like apoptosis, iron homeostasis and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V and VI) was regulated in response to tissue damage

Histone H4 lysine acetylation regulated by MOF (males absent on the first), was initially discovered as a dosage compensation epigenetic mark. Recent studies have revealed, however, that the epigenetic mark has a critical role in cellular function both during oogenesis as well as oncogenesis. Detailed molecular analysis of H4K16 isoforms and other post-translational modified histones has been limited by the lack of means to prepare sufficient material for in vitro study. This paper describes an improved method to prepare acetylated H4K16 as well as other covalently modified histone H4 isoform for biophysical studies.

The public dissemination of data is an integral part of the life sciences. In the field of proteomics too, data sharing has taken off over the last few years, with the first downstream uses of these data quickly gaining prominence. At the same time, the recent unfortunate demise of two repositories, NCBI Peptidome and ProteomeCommons Tranche, has shown the frailty of such data gathering efforts. Heroic efforts by the PRIDE and Peptidome teams to rescue the Peptidome data have now ensured their continued availability to the field, and alternatives have already been put in place for Tranche. But with public data increasingly at the hub of the life sciences, it is a good time to look at the proteomics data ecosystem in some more detail.

2D gel electrophoresis remains one of the main experimental approaches in proteome analysis. However, co-migration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification and PTM detection. We have optimized a third additional step of in-gel separation to alleviate co-migration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described.

An emerging approach for multiplexed targeted proteomics involves bottom-up LC-multiple-reaction monitoring (MRM)-MS, with stable isotope-labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer-biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM-based method. We have demonstrated excellent retention time reproducibility (median inter-day CV: 0.08%) and signal stability (median inter-day CV: 4.5% for the analytical platform and 6.1% for the bottom-up workflow) for the 27 biomarker proteins (represented by 57 interference-free peptides). The linear dynamic range for the MRM assays spanned four orders-of-magnitude, with 25 assays covering a 10³–10⁴ range in protein concentration. The lowest-abundance quantifiable protein in our biomarker panel was insulin-like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer-associated protein biomarker panels in human plasma, prior to clinical use.

The heat and ethanol stress response of Bacillus licheniformis DSM13 was analyzed at the transcriptional and/ or translational level. During heat shock, regulons known to be heat-induced in Bacillus subtilis 168 are upregulated in B. licheniformis, such as the HrcA, SigB, CtsR and CssRS regulon. Upregulation of the SigY regulon and of genes controlled by other ECF type sigma factors indicates a cell wall stress triggered by the heat shock. Furthermore, tryptophan synthesis enzymes were upregulated in heat stressed cells as well as regulons involved in usage of alternative carbon and nitrogen sources. Ethanol stress led to an induction of the SigB, HrcA and CtsR regulons. As indicated by the upregulation of a SigM-dependent protein, ethanol also triggered a cell wall stress. To characterize the SigB regulon of B. licheniformis, we analyzed the heat stress response of a sigB mutant. It is shown that the B. licheniformis SigB regulon comprises additional genes, some of which do not exist in B. subtilis such as BLi03885, encoding a hypothetical protein, the Na/solute symporter gene BLi02212, the arginase homolog-encoding gene BLi00198 and mcrA, encoding a protein with endonuclease activity.

Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague-Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and 2 groups were refed for one week with a standard diet supplemented with non-essential amino acids (NEAA group) or Cit- (CIT group). Results of the proteomic approach were validated using targeted western blot analysis and assessment of gene expression of the related-genes. Maximal activities of the key-enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituants and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs AL and R, p<0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs AL, p<0.05). However, maximal activities of key-enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs AL and R, p<0.05).. In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia towards anaerobia

Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics-mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well characterized plants, including the well-established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Towards an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) post translational modification of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.

Exosomes are small extracellular 40–100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist pre-metastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signalling molecules fundamental to tumour progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40–100 nm in diameter, were of a buoyant density ∼1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, EGFR, JAG1, SRC, TNIK) and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalisation of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in CRC cell exosomes. The selective enrichment of metastatic factors and signalling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the crosstalk between tumour and stromal cells in the tumour microenvironment.

We present new statistical approaches for identification of proteins with expression levels that are significantly changed when applying meta-analysis to two or more independent experiments. We showed that the Euclidean distance measure has reduced risk of false positives compared to the rank product method. Our Ψ-ranking method has advantages over the traditional fold-change approach by incorporating both the fold-change direction as well as the p-value. In addition, the second novel method, Π-ranking, considers the ratio of the fold-change and thus integrates all three parameters. We further improved the latter by introducing our third technique, Σ-ranking, which combines all three parameters in a balanced non-parametric approach.

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion (PE). Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study we report the first global proteomic analysis of highly purified MVs derived from human non-small-cell-lung-cancer (NSCLC) pleural effusion. Using nano-LC–MS/MS following 1-D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to EGFR signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains are associated with a satellite RNA (satRNA) that modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts, with and without PSV-P satRNA, and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared, 29 differentially regulated proteins were found, whereas 40 and 60 such proteins were found, respectively, when comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT. The presence of satRNA mostly decreased the amounts of the affected host proteins. 

Low root temperature causes a decrease in water uptake, which leads to mineral and nutrient deficiencies with potentially decreased root and shoot growth. Differential temperature effects in plants have been studied extensively, however, the effect of root chilling on the global protein expression in shoots has not been explored. In this study, we imposed chilling temperatures on roots of rice plants while maintaining shoots at optimum atmospheric temperature. Shoot materials (growing zones and leaves) were harvested at five points over a time course of four days, including a two-day recovery period. Proteins were quantified by tandem mass tags and triple stage mass spectrometry, using a method developed to overcome ratio compression in isobaric-labelled quantitation. Over 3000 proteins in each of the tissues were quantified by multiple peptides. Proteins significantly differentially expressed as compared with the control included ABA-responsive and drought associated proteins. The data also contained evidence of a possible induction of a sugar signalling pathway.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% (w/v) PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western-blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2-DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2-D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for down-stream proteomics analysis.

Acute myeloid leukemia (AML) is a heterogenous disease with differential oncogene association, outcome and treatment regimens. Treatment strategies for AML have improved outcome but despite increased molecular biological information AML is still associated with poor prognosis. Proteomic analysis on the effects of a range of leukemogenic oncogenes showed that the protein transglutaminase2 (TG2) is expressed at greater levels as a consequence of oncogenic transformation. Further analysis of this observation was performed with 511 AML samples using reverse phase proteomic arrays, demonstrating that TG2 expression was higher at relapse than diagnosis in many cases. In addition elevated TG2 expression correlated with increased expression of numerous adhesion proteins and many apoptosis regulating proteins, two processes related to leukemogenesis. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment.

O-GlcNAcylation is a dynamic posttranslational modification of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2-D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with 7 being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc modified proteins might be one of the vital malignant characteristics of cancer.

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study we aimed to identify Estrogen Receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in-vitro GST-Pull down assay with ERα ligand binding domain (ERα-LBD) and mass spectrometry based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for co-repressor interaction with ERα. We show that these two proteins physically interact with each other both in-vitro as well as in-vivo by GST-pull down and co-immunoprecipitation respectively. We further show that these two proteins also co-localize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrates that over expression of Profilin1 inhibits ERα mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 over expression in MCF7 cells leads to inhibition of cell proliferation which apparently is due to enhanced apoptosis. In nutshell, these data indicate that mass spectrometry based proteomics approach identifies a novel ERα interacting protein Profilin1 which serves as a putative co-repressor of ERα functions.

Fruit tree crops are agricultural commodities of high economic importance, while fruits also represent one of the most vital components of the human diet. Therefore, a great effort has been made to understand the molecular mechanisms covering fundamental biological processes in fruit tree physiology and fruit biology. Thanks to the development of cutting-edge ‘omics’ technologies such as proteomic analysis, scientists now have powerful tools to support traditional fruit tree research. Such proteomic analyses are establishing high-density 2-DE reference maps and peptide mass fingerprint databases that can lead fruit science into a new post-genomic research era. Here, an overview of the application of proteomics in key aspects of fruit tree physiology as well as in fruit biology, including defense responses to abiotic and biotic stress factors, is presented. Α panoramic view of ripening-related proteins is also discussed, as an example of proteomic application in fruit science.

Water-deficit or dehydration hampers plant growth and development, and shrinks harvest size of major crop species worldwide. Therefore, a better understanding of dehydration response is the key to decipher the regulatory mechanism of better adaptation. In recent years, nuclear proteomics has become an attractive area of research, particularly to study the role of nucleus in stress response. In this study, a proteome of dehydration-sensitive chickpea cultivar (ICCV-2) was generated from nuclei-enriched fractions. The LC-MS/MS analysis led to the identification of 75 differentially expressed proteins presumably associated with different metabolic and regulatory pathways. Nuclear localization of three candidate proteins was validated by transient expression assay. The ICCV-2 proteome was then compared with that of JG-62, a tolerant cultivar. The differential proteomics and in silico analysis revealed cultivar-specific differential expression of many proteins involved in various cellular functions. The differential tolerance could be attributed to altered expression of many structural proteins and the proteins involved in stress adaptation, notably the ROS catabolizing enzymes. Further, a comprehensive comparison on the abiotic stress-responsive nuclear proteome was performed using the datasets published thus far. These findings might expedite the functional determination of the dehydration-responsive proteins and their prioritization as potential molecular targets for better adaptation.

Seed germination is a critical process in the life cycle of higher plants. During germination, the imbibed mature seed is highly sensitive to different environmental factors. However, knowledge about the molecular and physiological mechanisms underlying the environmental effects on germination has been lacking. Recent proteomics work has provided invaluable insight into the molecular processes in germinating seeds of Arabidopsis, rice (Oryza sativa), soybean (Glycine max), barley (Hordeum vulgare), maize (Zea mays), tea (Camellia sinensis), European beech (Fagus sylvatica), and Norway maple (Acer platanoides) under different treatments including metal ions (e.g. copper and cadmium), drought, low temperature, hormones, and chemicals (gibberellic acid, abscisic acid, salicylic acid, and α-amanitin), as well as Fusarium graminearum infection. A total of 561 environmental factor-responsive proteins have been identified with various expression patterns in germinating seeds. The data highlight diverse regulatory and metabolic mechanisms upon seed germination, including induction of environmental factor-responsive signaling pathways, seed storage reserve mobilization and utilization, enhancement of DNA repair and modification, regulation of gene expression and protein synthesis, modulation of cell structure, and cell defense. In this review, we summarize the interesting findings and discuss the relevance and significance for our understanding of environmental regulation of seed germination.

Antibody arrays have been used as an effective method for simultaneously detecting multiple proteins such as cytokines. However, their use in quantifying a large number of cellular proteins has the following limitations: 1) unsuitable for simultaneously detecting proteins that may form complexes with each other; 2) incapable of quantitatively detecting more than one epitope of each protein such as phospho- and non-phospho epitopes; and 3) incapable of simultaneously detecting multiple biomarkers on solid surfaces such as formalin-fixed tissue sections. Using a novel multiple epitope detection (MED) technique, we have overcome these limitations and have improved upon currently available antibody-based protein detection technologies. The MED technique employs primary antibody detection of epitopes within fixed cells or tissue, followed by elution of bound antibodies, and subsequent quantification of the eluted antibodies by an epitope array. Using the MED method, we demonstrate accurate detection of individual proteins even in complex with each other, simultaneous quantitative detection of phospho- and non-phospho epitopes on a protein, and sensitive detection of multiple biomarkers on formalin-fixed tissue sections.

Exosomes are small microvesicles secreted from the late endosomal compartment of cells. Although an increasing body of evidence indicates that they play a pivotal role in cell-to-cell communication, the biological functions of exosomes are far from being fully understood. Recent work has revealed detailed proteomic profiles of exosomes from cell lines and body fluids, which may provide clues to understanding their biological significance and general importance in human diseases. Metalloproteinases include the cell surface-anchored sheddases ADAMs (a disintegrin and metalloproteinases), as well as cell surface-bound and soluble MMPs (matrix metalloproteinases) and these extracellular proteases have been detected in exosomes by proteomic analyses. Exosomes play a key role in the transfer of proteins to other cells and metalloproteinases may provide a novel platform where ectodomain shedding by these membrane proteases alters make up of the recipient cell's surface. This review aims to address some of the facets of exosome biology with particular emphasis on the proteolytic factors and we discuss their potential involvement in human diseases, especially tumor biology.

Plants are continually challenged to recognize and respond to adverse changes in their environment to avoid detrimental effects on growth and development. Understanding the mechanisms that crop plants employ to resist and tolerate abiotic stress is of considerable interest for designing agriculture breeding strategies to ensure sustainable productivity. The application of proteomics technologies to advance our knowledge in crop plant abiotic stress tolerance has increased dramatically in the past few years as evidenced by the large amount of publications in this area. This is attributed to advances in various technology platforms associated with MS-based techniques as well as the accessibility of proteomics units to a wider plant research community. This review summarizes the work which has been reported for major crop plants and evaluates the findings in context of the approaches that are widely employed with the aim to encourage broadening the strategies used to increase coverage of the proteome

Exosomes and microvesiscles (MVs) are nanometer sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)-associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC-derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.

N^ε-lysine acetylation, a reversible and highly regulated posttranslational modification, has been shown to occur in the model Gram-negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram-positive bacterium. Through anti-acetyllysine antibody-based immunoseparation of acetylpeptides followed by nano-HPLC/MS/MS analysis, we identified 332 unique lysine-acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. 59 of the lysine-acetylated proteins showed homology with lysine-acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH and YfmT, indicating that lysine acetylation may affect their activities. In 2-amino-3-ketobutyrate CoA ligase Kbl, a class II aminotransferase, a lysine residue involved in pyridoxal phosphate attachment was found to be acetylated. This dataset provides evidence for the generality of lysine acetylation in eubacteria and opens opportunities to explore the consequences of acetylation modification on the molecular physiology of B. subtilis.

Plants enhance their cold stress tolerance by cold acclimation, a process which results in vast reprogramming of transcriptome, proteome and metabolome. Evidence suggests nitric oxide (NO) production during cold stress which regulates genes (especially the C-repeat binding factor (CBF) cold stress signalling pathway), diverse proteins including transcription factors (TFs) and phosphosphingolipids. About 59% (redox), 50% (defence/stress) and 30% (signalling) cold responsive proteins are modulated by NO-based post translational modifications (PTMs) namely S-nitrosylation, tyrosine nitration and S-glutathionylation, suggesting a cross-talk between NO and cold. Analysis of cold stress responsive deep proteome in apoplast, mitochondria, chloroplast and nucleus suggested continuation of this cross-talk in sub-cellular systems. Modulation of cold responsive proteins by these PTMs right from cytoskeletal elements in plasma membrane to TFs in nucleus suggests a novel regulation of cold stress signalling. NO-mediated altered protein transport in nucleus seems an important stress regulatory mechanism. This review addresses the NO and cold stress signalling cross-talk to present the overview of this novel regulatory mechanism.

The rhizobium–legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene-inducing plant flavonoid naringenin (10 μM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein-coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including β(1–2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide-specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT-PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high-resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis-associated proteins, whose flavonoid-dependency had not yet been reported.

Tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the Tomato chlorotic mottle virus (ToCMoV). Virulence factor AC2 is considered crucial for a successful virus–plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an RNA silencing suppressor factor. However, the exact functions of the AC2 protein of the begomovirus ToCMoV are not yet established. The aim of the present study was to identify differentially expressed proteins of the model plant Nicotiana benthamiana in response to the expression of the AC2 gene, isolated from ToCMoV. N. benthamiana plants were inoculated with Agrobacterium tumefaciens containing the viral vector Potato virus X (PVX) and with the PVX-AC2 construction. 2DE was performed and proteins were identified by MS. The results showed that the expression of ToCMoV AC2 alters the levels of several host proteins, which are important for normal plant development, causing an imbalance in cellular homeostasis. This study highlights the effect of AC2 in the modulation of plant defense processes by increasing the expression of several oxidative stress-related and pathogenesis-related proteins, as well as its role in modulating the proteome of the photosynthesis and energy production systems.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, and that severely affects yield loss (upto 50%) of total rice production. Here, we report a proteomics investigation of Xoo (compatible race K3)-secreted proteins, isolated from its in vitro culture and in planta infected rice leaves. 2DE coupled with MALDI-TOF-MS and/or nLC-ESI-MS/MS approaches identified 139 protein spots (out of 153 differential spots), encoding 109 unique proteins. Identified proteins belonged to multiple biological and molecular functions. Metabolic and nutrient uptake proteins were common up to both in vitro and in planta secretomes. However, pathogenicity, protease/peptidase, and host defense-related proteins were highly or specifically expressed during in planta infection. A good correlation was observed between protein and transcript abundances for nine proteins secreted in planta as per semiquantitative RT-PCR analysis. Transgenic rice leaf sheath (carrying PBZ1 promoter::GFP cell death reporter), when used to express a few of the identified secretory proteins, showed a direct activation of cell death signaling, suggesting their involvement in pathogenicity related with secretion effectors. This work furthers our understanding of rice bacterial blight disease, and serves as a resource for possible translation in generating disease resistant rice plants for improved seed yield.

To screen target proteins of oxidative stress which mediate the effects of exercise on preventing nonalcoholic fatty liver disease (NAFLD), the methods for selecting carbonylated proteins were modified, and carbonylated proteins were profiled. The results showed that treadmill training reduced oxidative stress and the levels of intrahepatic triglyceride (IHTG). The changes in IHTG showed a significant positive correlation with oxidative stress as indicated by malondialdehyde level. Further results from proteomics illustrated that 17 functional proteins were susceptible to oxidative modification, and exercise protected three proteins from carbonylation. The latter three proteins may serve as both direct target proteins of oxidative stress and mediators contributing to the beneficial effects of exercise. In particular, a long-chain specific acyl-CoA dehydrogenase (ACADL) which was a key enzyme in lipid metabolism was not carbonylated and with higher activities in exercise group. These findings indicate that this modified technique is practical and powerful in selecting carbonylated proteins. Long-term treadmill training is effective in ameliorating oxidative stress and preventing the accumulation of IHTG. Among the 17 target proteins of oxidative modification, three proteins contribute to the beneficial effects of exercise. Preventing ACADL from carbonylation may be involved in the physiological mechanism of exercise-induced NAFLD improvement.

Genetic and microarray analyses have provided useful information in the area of plant and pathogen interactions. Pseudomonas syringae pv. tomato DC3000 (Pst) causes bacterial speck disease in tomato. Previous studies have shown that changes in response to pathogen infection at transcript level are variable at different time points. This study provides information not only on proteomic changes between a resistant and a susceptible genotype, but also information on changes between an early and a late time point. Using the iTRAQ quantitative proteomics approach, we have identified 2369 proteins in tomato leaves, and 477 of them were determined to be responsive to Pst inoculation. Unique and differential proteins after each comparison were further analyzed to provide information about protein changes and the potential functions they play in the pathogen response. This information is applicable not only to tomato proteomics, but also adds to the repertoire of proteins now available for crop proteomic analysis and how they change in response to pathogen infection.

Many histone covalent modifications have been identified and shown to play key regulatory roles in eukaryotic transcription, DNA damage repair, and replication. In vitro experiments designed to understand the mechanistic role of individual modifications require the availability of substantial quantities of pure histones, homogeneously modified at specific residues. We have applied the amber stop codon/suppressor tRNA strategy to the production of histone H4 acetylated at lysine 16, a particularly important isoform of this histone. Our success relies on adapting the H4 DNA sequence to the codon preference of E. coli and on preventing the premature decay of the H4 mRNA. These modifications to the original procedure render it easily applicable to the generation of any covalently modified histone H4 isoform.

A fully integrated platform was developed for capturing/fractionating human fucome from disease-free and breast cancer sera. It comprised a multicolumn operated by HPLC pumps and switching valves for the simultaneous depletion of high abundance proteins via affinity-based subtraction and the capturing of fucosylated glycoproteins via lectin affinity chromatography followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) were utilized. The platform allowed the “cascading” of the serum sample from column-to-column in the liquid phase with no sample manipulation between the various steps. This guaranteed no sample loss and no propagation of experimental biases between the various columns. Finally, the fucome was fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to trypsinolysis for LC-MS/MS analysis. This permitted the identification of the differentially expressed proteins (DEP) in breast cancer serum yielding a broad panel of 35 DEP from the combined LTA and AAL captured proteins and a narrower panel of eight DEP that were commonly differentially expressed in both LTA and AAL fractions, which are considered as more representative of cancer altered fucome.

Expressed prostatic secretions (EPS) are proximal fluids of the prostate that are increasingly being utilized as a clinical source for diagnostic and prognostic assays for prostate cancer (PCa). These fluids contain an abundant amount of microvesicles reflecting the secretory function of the prostate gland, and their protein composition remains poorly defined in relation to PCa. Using expressed prostatic secretions in urine (EPS-urine), exosome preparations were characterized by a shotgun proteomics procedure. In pooled EPS-urine exosome samples, ∼900 proteins were detected. Many of these have not been previously observed in the soluble proteome of EPS generated by our labs or other related exosome proteomes. We performed systematic comparisons of our data against previously published, prostate-related proteomes, and global annotation analyses to highlight functional processes within the proteome of EPS-urine derived exosomes. The acquired proteomic data have been deposited to the Tranche repository and will lay the foundation for more extensive investigations of PCa derived exosomes in the context of biomarker discovery and cancer biology.

Rice is an important cereal crop and has become a model monocot for research into crop biology. Rice seeds currently feed more than half of the world's population and the demand for rice seeds is rapidly increasing because of the fast-growing world population. However, the molecular mechanisms underlying rice seed development is incompletely understood. Genetic and molecular studies have developed our understanding of substantial proteins related to rice seed development. Recent advancements in proteomics have revolutionized the research on seed development at the single gene or protein level. Proteomic studies in rice seeds have provided the molecular explanation for cellular and metabolic events as well as environmental stress responses that occur during embryo and endosperm development. They have also led to the new identification of a large number of proteins associated with regulating seed development such as those involved in stress tolerance and RNA metabolism. In the future, proteomics, combined with genetic, cytological, and molecular tools, will help to elucidate the molecular pathways underlying seed development control and help in the development of valuable and potential strategies for improving yield, quality, and stress tolerance in rice and other cereals. Here, we reviewed recent progress in understanding the mechanisms of seed development in rice with the use of proteomics.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that can self-renew, metastasize, and promote cancer recurrence. A comprehensive characterization of the CSC proteome has been hampered due to their scarcity and rapid differentiation. Here, we present a systematic analysis of the cell-surface proteome using a CSC-like cell line derived from MDA-MB453 breast cancer cells, which exhibited a CD44⁺/CD24⁻ (where CD is cluster of differentiation) phenotype and chemoresistance. We identified differentially expressed proteins in CSC-like cells, including upregulated plasma membrane proteins such as CD44, CD133, epidermal growth factor receptor (EGFR), CD147, cadherin 1, integrins, and catenin (cadherin-associated protein), beta 1 (CTNNB1), using an in-situ biotinylation approach followed by MS analysis. We examined the role of CD147 in the promotion of CSC growth and survival, and demonstrated that inhibition of CD147 with a monoclonal antibody induced significant inhibition of cell growth. siRNA-mediated silencing of CD147 gene expression restored the sensitivity of CSC-like cells to 5-fluorouracil (5-FU), along with decreasing the expression of thymidylate synthase, p-AKT, and β-catenin, while increasing the expression of p-glycogen synthase kinase (GSK)3β. Increased CD147 expression in the CSC-like cells, as seen by proteomic analysis, and the functional consequences of CD147 overexpression in CSC-like cells suggest that CD147 may be one of the critical cell-surface proteins involved in promoting chemoresistance and survival in CSCs.

Extracellular vesicles (EVs), including exosomes, act as biological effectors and as carriers of oncogenic signatures in human cancer. The molecular composition and accessibility of EVs in biofluids open unprecedented diagnostic opportunities in malignancies where tumour tissue is difficult to sample, especially in primary and metastatic brain tumours. The ongoing genetic discovery of driver mutations defines the ever increasing numbers of distinct molecular subtypes of brain tumours (orphan diseases), a complexity that may soon be translated into alterations in functional proteins and their oncogenic networks. This may likely be extended to real time changes engendered by the disease progression, tumour heterogeneity, inter-individual variations and therapeutic responses. Meeting these challenges through EV analysis is dependent on technological progress in such areas as generation of mutation- and phospho-specific antibodies, antibody array platforms, nanotechnology, microfluidics, NMR spectroscopy, MS and MRM approaches of quantitative proteomics, which should not be underestimated. Still, vesiculation emerges as a unique process that could be harnessed for the benefit of more individualised patient care.

The exosome is a secreted microvesicle that has been shown to contain genetic material and proteins and is involved in multiple levels of cellular communication. The cardiovascular exosome proteome is a promising subproteome that warrants investigation since a detailed understanding of its role in the heart should improve our comprehension of intercellular communication in the heart, and may even assist in biomarker discovery. Indeed, uncovering the role of the exosome in cardiovascular physiology could be accomplished with the application of scientific approaches and insights gained from studies of exosomes in other fields, such as cancer biology and immunology, where much of the established knowledge of the exosome has been generated. In the present review, we discuss the relevant literature and examine areas of investigation that would bring the cardiovascular exosome to the forefront of intercellular communication in the heart.

The PRIDE database, developed and maintained at the European Bioinformatics Institute (EBI), is one of the most prominent data repositories dedicated to high throughput MS-based proteomics data. Peptidome, developed by the National Center for Biotechnology Information (NCBI) as a sibling resource to PRIDE, was discontinued due to funding constraints in April 2011. A joint effort between the two teams was started soon after the Peptidome closure to ensure that data were not “lost” to the wider proteomics community by exporting it to PRIDE. As a result, data in the low terabyte range have been migrated from Peptidome to PRIDE and made publicly available under experiment accessions 17 900–18 271, representing 54 projects, ∼53 million mass spectra, ∼10 million peptide identifications, ∼650 000 protein identifications, ∼1.1 million biologically relevant protein modifications, and 28 species, from more than 30 different labs.

To guarantee sufficient food supply for a growing world population, efforts towards improving crop yield and plant resistance should be complemented with efforts to reduce post-harvest losses. Post-harvest losses are substantial and occur at different stages of the food chain in developed and developing countries. In recent years, a substantially increasing interest can be seen in the application of proteomics to understand post-harvest events. In the near future post-harvest proteomics will be poised to move from fundamental research to aiding the reduction of food losses. Proteomics research can help in reducing food losses through (i) identification and validation of gene products associated to specific quality traits supporting marker-assisted crop improvement programmes, (ii) delivering markers of initial quality that allow optimisation of distribution conditions and prediction of remaining shelf-life for decision support systems and (iii) delivering early detection tools of physiological or pathogen-related post-harvest problems. In this manuscript, recent proteomics studies on post-harvest and stress physiology are reviewed and discussed. Perspectives on future directions of post-harvest proteomics studies aiming to reduce food losses are presented.

To understand early events in plant–pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins that help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium head blight on wheat, a serious cereal disease found in many cereal-growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative MS to compare the secretomes of wild-type with two nonpathogenic deletion mutants of F. graminearum, Δtri6, and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins that decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins, and proteins of unknown function.

Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed.

Exosomes are nanometer-sized vesicles (40–100 nm diameter) of endocytic origin released from different cell types under both normal and pathological conditions. They function as cell free messengers, playing a relevant role in the cell–cell communication that is strongly related to the nature of the molecules (proteins, mRNAs, miRNAs, and lipids) that they transport. Tumor cells actively shed exosomes into their surrounding microenvironment and growing evidence indicates that these vesicles have pleiotropic functions in the regulation of tumor progression, promoting immune escape, tumor invasion, neovascularization, and metastasis. During the last few years remarkable efforts have been made to obtain an accurate definition of the protein content of tumor-derived exosomes (TDEs) by applying MS-based proteomic technologies. To date, TDEs proteomic studies have been mainly utilized to catalog TDEs proteins with the purpose of identifying disease biomarkers. The future challenge for improving our understanding and characterization of TDEs will be the implementation of new systems-driven and proteomic integrative strategies. The aim of this article is to provide an overview of the most characterized exosomes-mediated mechanisms that contribute to the pathogenesis of cancer and to review recent proteomics data that support the protumorigenic role of TDEs.

Mammalian cells secrete two types of extracellular vesicles either constitutively or in a regulated manner: exosomes (50–100 nm in diameter) released from the intracellular compartment and ectosomes (also called microvesicles, 100–1000 nm in diameter) shed directly from the plasma membrane. Extracellular vesicles are bilayered proteolipids enriched with proteins, mRNAs, microRNAs, and lipids. In recent years, much data have been collected regarding the specific components of extracellular vesicles from various cell types and body fluids using proteomic, transcriptomic, and lipidomic methods. These studies have revealed that extracellular vesicles harbor specific types of proteins, mRNAs, miRNAs, and lipids rather than random cellular components. These results provide valuable information on the molecular mechanisms involved in vesicular cargo-sorting and biogenesis. Furthermore, studies of these complex extracellular organelles have facilitated conceptual advancements in the field of intercellular communication under physiological and pathological conditions as well as for disease-specific biomarker discovery. This review focuses on the proteomic, transcriptomic, and lipidomic profiles of extracellular vesicles, and will briefly summarize recent advances in the biology, function, and diagnostic potential of vesicle-specific components.

Extracellular vesicles (EVs), membrane vesicles that are secreted by a variety of mammalian cell types, have been shown to play an important role in intercellular communication. The contents of EVs, including proteins, microRNAs, and mRNAs, vary according to the cell type that secreted them. Accordingly, researchers have demonstrated that EVs derived from various cell types play different roles in biological phenomena. Considering the ubiquitous presence of mesenchymal stem cells (MSCs) in the body, MSC-derived EVs may take part in a wide range of events. In particular, MSCs have recently attracted much attention due to the therapeutic effects of their secretory factors. MSC-derived EVs may therefore provide novel therapeutic approaches. In this review, we first summarize the wide range of functions of EVs released from different cell types, emphasizing that EVs echo the phenotype of their parent cell. Then, we describe the various therapeutic effects of MSCs and pay particular attention to the significance of their paracrine effect. We then survey recent reports on MSC-derived EVs and consider the therapeutic potential of MSC-derived EVs. Finally, we discuss remaining issues that must be addressed before realizing the practical application of MSC-derived EVs, and we provide some suggestions for enhancing their therapeutic efficiency.

Over the last decade, there has been increasing research interest in urinary exosomes and their relationship with kidney physiology and disease. Protocols for isolating urinary exosomes have been refined and the exosomal proteome has been extensively catalogued and reported to contain proteins from the kidney's glomerulus and all sections of the nephron. In animal and human biomarker discovery studies, this proteome changes to reflect the underlying pathophysiology of certain kidney diseases. In addition to proteins, exosomes from urine have been demonstrated to contain RNA species, another new reservoir for biomarker discovery. Exosomes have the capacity to shuttle their cargo between kidney cells and change the recipient cell's proteome and function, and may represent a mechanism for cell-to-cell signalling along the nephron. Significant challenges remain; methods for urinary exosome collection need optimisation if “real-life” clinical utility is to be achieved, consensus is needed regarding normalisation of changes in exosomal protein and RNA, larger scale exosome biomarker validation studies remain to be performed, and whether exosomes signal between cells in vivo remains an intriguing, but untested, hypothesis.

While the genome represents a static moiety, the proteome is more dynamic and can change in response to stimuli. As such, proteomics may reveal the effect of various drugs on cellular mechanisms. Protein alterations upon exogenous perturbations are vital in outlining species-to-species differences that cannot otherwise be measured quantitatively. Specifically, nicotine has been shown to be an independent risk factor for a multitude of diseases. In pancreatic research its mechanism of action remains unresolved. The pioneering work of Paulo et al. (Proteomics 2013, 13, 1499–1512) is a major step toward understanding the role of nicotine, a principal toxin in cigarette smoke, in pancreatic disease. Equally important, Paulo et al. examine the effect of nicotine on stellate cells across three species, demonstrating the importance of identifying species-specific effects in translational research.

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator^TM DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500–5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.

The use of MS for characterization of small molecules, nucleotides, and proteins in model organisms as well as primary tissues and clinical samples continues to proliferate at a rapid pace. The complexity and dynamic range of target analytes in biological systems hinders comprehensive analysis and simultaneously drives improvements in instrument hardware and software. As a result, state-of-the-art commercial mass spectrometers are equipped with sophisticated embedded control systems that provide robust acquisition methods accessed through intuitive graphical interfaces. Although optimized for speed, these preconfigured scan functions are otherwise closed to end-user customization beyond simple, analytical-centric parameters supplied by the manufacturer. Here, we present an open-source framework (mzAPI/Live) that enables users to generate arbitrarily complex LC-MSⁿ acquisition methods via simple Python scripting. As a powerful proof-of-concept, we demonstrate real-time assignment of tandem mass spectra through rapid query of NIST peptide libraries. This represents an unprecedented capability to make acquisition decisions based on knowledge of analyte structures determined during the run itself, thus providing a path toward biology-driven MS data acquisition for the broader community.

Simple protein separation by 1DE is a widely used method to reduce sample complexity and to prepare proteins for mass spectrometric identification via in-gel digestion. While several automated solutions are available for in-gel digestion particularly of small cylindric gel plugs derived from 2D gels, the processing of larger 1D gel-derived gel bands with liquid handling work stations is less well established in the field. Here, we introduce a digestion device tailored to this purpose and validate its performance in comparison to manual in-gel digestion. For relative quantification purposes, we extend the in-gel digestion procedure by iTRAQ labeling of the tryptic peptides and show that automation of the entire workflow results in robust quantification of proteins from samples of different complexity and dynamic range. We conclude that automation improves accuracy and reproducibility of our iTRAQ workflow as it minimizes the variability in both, digestion and labeling efficiency, the two major causes of irreproducible results in chemical labeling approaches.

In the present work, we report a novel on-target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on-tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample-handling steps and time are the features that make this method appealing to the many laboratories working with high-throughput sample treatment.

Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS and BRAF mutation status was determined in 172 ACF identified in the colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology (serrated vs. distended). Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) microdissection with an ultrasensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of posttranslational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and ERK-2 were quantified in samples containing as little as 16 ng of total protein recovered from <200 cells. ERK activation is responsible for observed hyperplasia found in these early lesions, but is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions and highlights the advantage of this approach over classical immunohistochemistry-based analyses.

High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K_D) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (K_D ∼ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (K_D ∼ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays.

Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies.

High throughput MS-based proteomic experiments generate large volumes of complex data and necessitate bioinformatics tools to facilitate their handling. Needs include means to archive data, to disseminate them to the scientific communities, and to organize and annotate them to facilitate their interpretation. We present here an evolution of PROTICdb, a database software that now handles MS data, including quantification. PROTICdb has been developed to be as independent as possible from tools used to produce the data. Biological samples and proteomics data are described using ontology terms. A Taverna workflow is embedded, thus permitting to automatically retrieve information related to identified proteins by querying external databases. Stored data can be displayed graphically and a “Query Builder” allows users to make sophisticated queries without knowledge on the underlying database structure. All resources can be accessed programmatically using a Java client API or RESTful web services, allowing the integration of PROTICdb in any portal. An example of application is presented, where proteins extracted from a maize leaf sample by four different methods were compared using a label-free shotgun method. Data are available at http://moulon.inra.fr/protic/public. PROTICdb thus provides means for data storage, enrichment, and dissemination of proteomics data.

Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.

As one of the most important post-translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N-linked glycans were identified by LC-ESI-MS/MS and MALDI-TOF/TOF-MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty-eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N-linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco-antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.

Toxic compounds in tobacco, such as nicotine, may adversely affect pancreatic function. We aim to determine nicotine-induced protein alterations in pancreatic cells, thereby revealing links between nicotine exposure and pancreatic disease. We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat, and human stellate cells and human duct cells) using MS-based techniques, specifically SDS-PAGE (gel) coupled with LC-MS/MS and spectral counting. We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine-treated or untreated cells. Interspecies comparisons of stellate cell proteins revealed several differentially abundant proteins (in nicotine treated versus untreated cells) common among the three species. Proteins appearing in all nicotine-treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B, and toll-interacting protein. Proteins that were differentially expressed upon nicotine treatment across cell lines were enriched in certain pathways, including nicotinic acetylcholine receptor, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease.

The filamentous fungus Aspergillus flavus is an opportunistic soil-borne pathogen that produces aflatoxins, the most potent naturally occurring carcinogenic compounds known. This work represents the first gel-based profiling analysis of A. flavus proteome and establishes a 2D proteome map. Using 2DE and MALDI-TOF-MS/MS, we identified 538 mycelial proteins of the aflatoxigenic strain NRRL 3357, the majority of which were functionally annotated as related to various cellular metabolic and biosynthetic processes. Additionally, a few enzymes from the aflatoxin synthesis pathway were also identified.

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β-secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter-aided sample preparation, strong-anion exchange fractionation, and label-free quantification. We used bace1-deficient zebrafish and quantified differences in protein levels between wild-type and bace1 −/− zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild-type and bace1 −/− zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty-four membrane proteins accumulated in the bace1 −/− brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican-1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock-out zebrafish tissue and demonstrates that combining gene knock-out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

Variation in water-holding capacity (WHC), which presents a major economic burden to the swine industry, is considered to be underpinned by variation at a molecular and biochemical level. High-resolution 2D DIGE followed by MS analysis and Western blot were used to unravel the proteome of muscle exudate, collected following centrifugation, in the pH 4–7 range. A first 2DE-based protein map of this substrate was produced where 89 spots were successfully characterised. Two phenotypes divergent for WHC plus one intermediate were compared with a view to deciphering the biochemical processes impacting on variation in WHC. Twenty spots were observed to be altered across the phenotypes. Of these, 14 represented sixteen proteins including metabolic enzymes, stress response proteins and structural proteins. Triosephosphate isomerase and transferrin showed a major difference between the two extreme phenotypes, and may have potential as biological markers for WHC prediction. Several members of the HSPs family were highlighted. This proteomic study makes an important contribution towards a more detailed molecular view of the processes behind WHC and will provide a valuable resource for future investigations.