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To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such labelfree quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by onedimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNF␣, interferon ␥, and IL1␤, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015230, 527-539, 2012.With recent advances in mass spectrometry, label-free quantitative proteomic approaches have progressed and are now considered as reliable and efficient methods to study protein expression level changes in complex mixtures. These approaches, which have been reviewed recently (1, 2), are based on the measurement either of the MS/MS sampling rate of a particular peptide or of its MS chromatographic peak area, these values being directly related to peptide abundance. The increase of instrument sequencing speed has benefited MS/MS spectral counting approaches by improving MS/MS sampling of peptide mixtures, whereas the introduction of high resolution analyzers such as FT-Orbitrap has boosted the use of methods based on peptide intensity measurements by greatly facilitating the matching of peptide peaks in different complex maps acquired independently. The most obvious advantage of these methods over isotopic labeling techniques is their ease of use at the sample preparation step, because they do not require any preliminary treatment to introduce a label into peptides or proteins. Being more straightforward, they also do not present the classical drawbacks of labeling methods, i.e., cost, applicability to limited types of samples (mostly cultured cells in the case of metabo...

Insulin-like growth factor-1 (IGF-1) and pituitary adenylyl cyclase activating polypeptide (PACAP) are both potent neurotrophic and antiapoptotic factors, which exert their effects via phosphorylation cascades initiated by tyrosine kinase and G-protein-coupled receptors, respectively. Here, we have adapted a recently described phosphoproteomic approach to neuronal cultures to characterize the phosphoproteomes generated by these neurotrophic factors. Unexpectedly, IGF-1 and PACAP increased the phosphorylation state of a common set of proteins in neurons. Using PACAP type 1 receptor (PAC1R) null mice, we showed that IGF-1 transactivated PAC1Rs constitutively associated with IGF-1 receptors. This effect was mediated by Src family kinases, which induced PAC1R phosphorylation on tyrosine residues. PAC1R transactivation was responsible for a large fraction of the IGF-1-associated phosphoproteome and played a critical role in the antiapoptotic activity of IGF-1. Hence, in contrast to the general opinion that the trophic activity of IGF-1 is solely mediated by tyrosine kinase receptor-associated signalling, we show that it involves a more complex signalling network dependent on the PAC1 Gs-protein-coupled receptor in neurons.

Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 μg/kg bw per day has been derived from an oral Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.acute toxicity study in mice. Based on this value and a large shellfish meat portion size of 400g, a concentration lower than 23 μg PnTX G/kg shellfish meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for shellfish production and identified data gaps to refine health risk assessment. Highlights► A state of knowledge of PnTXs and Vulcanodinium rugosum is proposed. ► A provisional acute healthbased guidance value for PnTX G of 0.13 μg/kg bw per day has been derived. ► PnTXs in shellfish should not exceed 23 μg PnTX G/kg. ► PnTXs should be included in national official monitoring programs for shellfish production. ► This work is a major step forward in taking into account this emerging hazard.

The implication of the cyanotoxin β-N-methylamino-l-alanine (BMAA) in long-lasting neurodegenerative disorders is still a matter of controversy. It has been alleged that chronic ingestion of BMAA through the food chain could be a causative agent of amyotrophic lateral sclerosis (ALS) and several related pathologies including Parkinson syndrome. Both in vitro and in vivo studies of the BMAA mode of action have focused on different molecular targets, demonstrating its toxicity to neuronal cells, especially motoneurons, and linking it to human neurodegenerative diseases. Historically, the hypothesis of BMAA-induced excitotoxicity following the stimulation of glutamate receptors has been established. However, in this paradigm, most studies have shown acute, rather than chronic effects of BMAA. More recently, the interaction of this toxin with neuromelanin, a pigment present in the nervous system, has opened a new research perspective. The issues raised by this toxin are related to its kinetics of action, and its possible incorporation into cellular proteins. It appears that BMAA neurotoxic activity involves different targets through several mechanisms known to favour the development of neurodegenerative processes.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 g). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins. Molecular & Cellular Proteomics 4:1085-1094, 2005.Since the last decade, MS has become one of the most powerful techniques in proteomics. Two-dimensional (2-D) 1 PAGE in combination with MALDI-TOF analysis provides a sensitive method for protein identification. An important challenge in proteomics deals with quantification of relative expression levels of individual proteins in two different biological samples. In this context, quantification by mass spectrometry has recently become a major alternative strategy to analysis based on densitometric or fluorescence technologies (1-4). Despite extensive work accomplished in the field of quantification, only few strategies have been associated with the widely used 2-D PAGE/MALDI.Quantification by mass spectrometry requires the production of differentially labeled peptides from two sets of proteins. The difference in expression levels is obtained by measuring the relative intensities of MS signals of the mixed labeled peptides. Different strategies have already been described (for reviews, see Refs. 2 and 5-8). These strategies differ in 1) the pre-or postdigestion labeling, 2) the choice of the labeled function (thiol, primary amine, or carboxylic acid), 3) the use of stable isotopes or alternative chemicals, and 4) the type of MS analysis (LC-MS/MS or MALDI-TOF MS). Predigestion labeling allows for greater quantification accuracy than postdigestion labeling. Indeed introducing the chemical modification early in the experimental procedure reduces differential loss of proteins during further biochemical steps and guarantees accuracy of the quantification. Predigestion labeling often involves stable isotope incorporation through either metabolic labeling or chemical labeling on a specific residue of proteins. Metabolic labeling, such as the stable isotope labeling by amino acids in cell culture (SILAC) method, is introduced in vivo ear...

Background: Target identification on tumor microvascularization is an opportunity for cancer therapy. Results: Membrane and secreted glycoproteins were identified on endothelial cells under hypoxia, and their function was assessed in zebrafish. Conclusion: Three novel hypoxia-regulated glycoproteins involved in angiogenesis have been identified and validated. Significance: We describe an approach that can be used to rapidly identify novel angiogenesis-related genes and their protein products.

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