Robust High-Throughput Proteomics Identification and Deamidation Quantitation of Extinct Species up to Pleistocene with Ultrahigh-Resolution MALDI-FTICR Mass Spectrometry.

Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.

Passages in culture and stimulation conditions influence protein expression of primary fibroblasts.

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-ß1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-ß1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-ß1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-ß1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using "omics" approaches.

Wild Wheat Rhizosphere-Associated Plant Growth-Promoting Bacteria Exudates: Effect on Root Development in Modern Wheat and Composition.

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur.

PDMS Curing Inhibition on 3D-Printed Molds: Why? Also, How to Avoid It?

Three-dimensional (3D)-printing techniques such as stereolithography (SLA) are currently gaining momentum for the production of miniaturized analytical devices and molds for soft lithography. However, most commercially available SLA resins inhibit polydimethylsiloxane (PDMS) curing, impeding reliable replication of the 3D-printed structures in this elastomeric material. Here, we report a systematic study, using 16 commercial resins, to identify a fast and straightforward treatment of 3D-printed structures and to support accurate PDMS replication using UV and/or thermal post-curing. In-depth analysis using Raman spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry revealed that phosphine oxide-based photo-initiators, leaching out of the 3D-printed structures, are poisoning the Pt-based PDMS catalyst. Yet, upon UV and/or thermal treatments, photo-initiators were both eliminated and recombined into high molecular weight species that were sequestered in the molds.

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex.

The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα-interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.

Regio- and Stereo-Specific Chemical Depolymerization of High Molecular Weight Polybutadiene and Polyisoprene for Their Analysis by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Comparison with Pyrolysis-Comprehensive Two-Dimensional Gas Chromatography/Mass Spectrometry, Atmospheric Solid Analysis Probe, Direct Inlet Probe-Atmospheric Pressure Chemical Ionization Mass Spectrometry, and Ion Mobility Spectrometry-Mass Spectrometry.

Polybutadiene (PB) and polyisoprene (PI), the two most common polydienes (PD), are involved in a large number of materials and used in a wide variety of applications. The characterization of these polymers by mass spectrometry (MS) continues to be very challenging due to their high insolubility and the difficulty to ionize them. In this work, a cross-metathesis reaction was used to generate end-functionalized acetoxy ionizable oligomers for the structural deciphering of different commercial PB and PI samples. A cross-metathesis reaction was carried out between polymers and the Z-1,4-diacetoxy-2-butene as a chain transfer agent in dichloromethane using a Hoveyda-Grubbs second-generation catalyst. Well-defined acetoxy telechelic structures were obtained and analyzed by Fourier transform ion cyclotron resonance (FTICR) high-resolution MS. However, after depolymerization, low molar mass polyolefins contained some units with different configurations, suggesting an olefin isomerization reaction due to the decomposition of the catalyst. The addition of an electron-deficient reagent such as 2,6-dichloro-1,4-benzoquinone suppressed this isomerization in the case of both Z- and E-PB and PI. Ion mobility spectrometry-mass spectrometry (IMS-MS) and energy-resolved tandem mass spectrometry (ERMS) analyses confirmed a successful isomerization suppression. For comparing the results obtained by depolymerization with classical methods for polymer analysis, pyrolysis-comprehensive two-dimensional gas chromatography/mass spectrometry (Py-GC × GC-MS), atmospheric solid analysis probe (ASAP), and direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) analyses were performed on the same polymers. This strategy can be applied on a variety of synthetic and natural not yet characterized polymers.

Heteroleptic Ruthenium(II) Complexes with Bathophenanthroline and Bathophenanthroline Disulfonate Disodium Salt as Fluorescent Dyes for In-Gel Protein Staining.

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent RuII tris(bathophenanthroline disulfonate) complex (Ru(BPS)3), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic RuII complexes Ru(BPS)2(BP) and Ru(BPS)(BP)2 containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS)3. It was shown by fluorescent and UV-vis measurements that novel RuII complexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP)2 can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs).

Palladium-Catalyzed Cross-Coupling of Gem-Bromofluoroalkenes with Alkylboronic Acids for the Synthesis of Alkylated Monofluoroalkenes.

Monofluoroalkenes are versatile fluorinated synthons in organic synthesis, medicinal chemistry and materials science. In light of the importance of alkyl-substituted monofluoroalkenes efficient synthesis of these moieties still represents a synthetic challenge. Herein, we described a mild and efficient methodology to obtain monofluoroalkenes through a stereospecific palladium-catalyzed alkylation of gem-bromofluoroalkenes with primary and strained secondary alkylboronic acids under mild conditions. This novel strategy gives access to a wide range of functionalized tri- and tetrasubstituted monofluoroalkenes in high yield, with good functional group tolerance, independently from the gem-bromofluoroalkenes geometry.

Two-dimensional mass spectrometry: new perspectives for tandem mass spectrometry.

Fourier transform ion cyclotron resonance mass analysers (FT-ICR MS) can offer the highest resolutions and mass accuracies in mass spectrometry. Mass spectra acquired in an FT-ICR MS can yield accurate elemental compositions of all compounds in a complex sample. Fragmentation caused by ion-neutral, ion-electron, or ion-photon interactions leads to more detailed structural information on compounds. The most often used method to correlate compounds and their fragment ions is to isolate the precursor ions from the sample before fragmentation. Two-dimensional mass spectrometry (2D MS) offers a method to correlate precursor and fragment ions without requiring precursor isolation. 2D MS therefore enables easy access to the fragmentation patterns of all compounds from complex samples. In this article, the principles of FT-ICR MS are reviewed and the 2D MS experiment is explained. Data processing for 2D MS is detailed, and the interpretation of 2D mass spectra is described.

Comparative Proteomic and Transcriptomic Analysis of Follistatin-Induced Skeletal Muscle Hypertrophy.

Skeletal muscle, the most abundant body tissue, plays vital roles in locomotion and metabolism. Myostatin is a negative regulator of skeletal muscle mass. In addition to increasing muscle mass, Myostatin inhibition impacts muscle contractility and energy metabolism. To decipher the mechanisms of action of the Myostatin inhibitors, we used proteomic and transcriptomic approaches to investigate the changes induced in skeletal muscles of transgenic mice overexpressing Follistatin, a physiological Myostatin inhibitor. Our proteomic workflow included a fractionation step to identify weakly expressed proteins and a comparison of fast versus slow muscles. Functional annotation of altered proteins supports the phenotypic changes induced by Myostatin inhibition, including modifications in energy metabolism, fiber type, insulin and calcium signaling, as well as membrane repair and regeneration. Less than 10% of the differentially expressed proteins were found to be also regulated at the mRNA level but the Biological Process annotation, and the KEGG pathways analysis of transcriptomic results shows a great concordance with the proteomic data. Thus this study describes the most extensive omics analysis of muscle overexpressing Follistatin, providing molecular-level insights to explain the observed muscle phenotypic changes.

Nonuniform Sampling Acquisition of Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Increased Mass Resolution of Tandem Mass Spectrometry Precursor Ions.

Obtaining the full MS/MS map for fragments and precursors of complex mixtures without hyphenation with chromatographic separation by a data-independent acquisition is a challenge in mass spectrometry which is solved by two-dimensional (2D) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Until now 2D FTICR MS afforded only a moderate resolution for precursor ion since this resolution is limited by the number of evolution interval steps to which the number of scans, the acquisition time, and the sample consumption are proportional. An overnight acquisition is already required to reach a quadrupole mass filter-like unit mass resolution. Here, we report that 2D FTICR MS using nonuniform sampling (NUS) obtained by randomly skipping points in the first dimension corresponding to the precursor selection gives access, after data processing, to the same structural information contained in a complex mixture. The resolution increases roughly as the inverse of the NUS ratio, up to 26 times at NUS 1/32, leading to an acquisition time reduced in the same ratio compared to a classical acquisition at the same resolution. As an example, the analysis of a natural oil is presented.

Efficient denoising algorithms for large experimental datasets and their applications in Fourier transform ion cyclotron resonance mass spectrometry.

Modern scientific research produces datasets of increasing size and complexity that require dedicated numerical methods to be processed. In many cases, the analysis of spectroscopic data involves the denoising of raw data before any further processing. Current efficient denoising algorithms require the singular value decomposition of a matrix with a size that scales up as the square of the data length, preventing their use on very large datasets. Taking advantage of recent progress on random projection and probabilistic algorithms, we developed a simple and efficient method for the denoising of very large datasets. Based on the QR decomposition of a matrix randomly sampled from the data, this approach allows a gain of nearly three orders of magnitude in processing time compared with classical singular value decomposition denoising. This procedure, called urQRd (uncoiled random QR denoising), strongly reduces the computer memory footprint and allows the denoising algorithm to be applied to virtually unlimited data size. The efficiency of these numerical tools is demonstrated on experimental data from high-resolution broadband Fourier transform ion cyclotron resonance mass spectrometry, which has applications in proteomics and metabolomics. We show that robust denoising is achieved in 2D spectra whose interpretation is severely impaired by scintillation noise. These denoising procedures can be adapted to many other data analysis domains where the size and/or the processing time are crucial.

Two-Dimensional Mass Spectrometry for Proteomics, a Comparative Study with Cytochrome c.

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows the correlation between precursor and fragment ions in tandem mass spectrometry without the need to isolate the precursor ion beforehand. 2D FT-ICR MS has been optimized as a data-independent method for the structural analysis of compounds in complex samples. Data processing methods and denoising algorithms have been developed to use it as an analytical tool. In the present study, the capabilities of 2D FT-ICR MS are explored with a tryptic digest of cytochrome c with both ECD and IRMPD as fragmentation modes. The 2D mass spectra showed useful fragmentation patterns of peptides over a dynamic range of almost 400. By using a quadratic calibration, fragment ion peaks could be successfully assigned. The correlation between precursor and fragment ions in the 2D mass spectra was more accurate than in MS/MS spectra after quadrupole isolation, due to the limitations of quadrupole isolation. The use of the second dimension allowed for successful fragment assignment from precursors that were separated by only m/z 0.0156. The resulting cleavage coverage of cytochrome c almost matched data provided by high-resolution FT-ICR MS/MS analysis, but the 2D FT-ICR MS method required only one experimental scan.

Theory for spiralling ions for 2D FT-ICR and comparison with precessing magnetization vectors in 2D NMR.

Two-dimensional (2D) Fourier transform ion cyclotron resonance (FT-ICR) offers an approach to mass spectrometry (MS) that pursuits similar objectives as MS/MS experiments. While the latter must focus on one ion species at a time, 2D FT ICR can examine all possible correlations due to ion fragmentation in a single experiment: correlations between precursors, charged and neutral fragments. We revisited the original 2D FT-ICR experiment that has hitherto fallen short of stimulating significant analytical applications, probably because it is technically demanding. These shortcomings can now be overcome by improved FT-ICR instrumentation and computer hard- and software. We seek to achieve a better understanding of the intricacies of the behavior of ions during a basic two-dimensional ICR sequence comprising three simple monochromatic pulses. Through simulations based on Lorentzian equations, we have mapped the ion trajectories for different pulse durations and phases.

2D fibrillar osteoid niche mimicry through inclusion of visco-elastic and topographical cues in gelatin-based networks.

Given the clinical need for osteoregenerative materials incorporating controlled biomimetic and biophysical cues, a novel highly-substituted norbornene-modified gelatin was developed enabling thiol-ene crosslinking exploiting thiolated gelatin as cell-interactive crosslinker. Comparing the number of physical crosslinks, the degree of hydrolytic degradation upon modification, the network density and the chemical crosslinking type, the osteogenic effect of visco-elastic and topographical properties was evaluated. This novel network outperformed conventional gelatin-based networks in terms of osteogenesis induction, as evidenced in 2D dental pulp stem cell seeding assays, resulting from the presentation of both a local (substrate elasticity, 25-40 kPa) and a bulk (compressive modulus, 25-45 kPa) osteogenic substrate modulus in combination with adequate fibrillar cell adhesion spacing to optimally transfer traction forces from the fibrillar ECM (as evidenced by mesh size determination with the rubber elasticity theory) and resulting in a 1.7-fold increase in calcium production (compared to the gold standard gelatin methacryloyl (GelMA)).

Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species.

The disulfide bond between cysteine 10 and cysteine 34 is required for CCL18 activity.

Asthma is a Th2-mediated disease that involves Th2 cell and eosinophil migration into the bronchial mucosa which is dependent upon the expression of a specific set of chemokines within the lung. Among them, CCL18 seems to play a key role because of its preferential expression in the lung, and its up-regulation by Th2 cytokines. Here, we show that the optimal naïve T cell and basophil chemotaxis, and basophil histamine release induced by rhCCL18 occurred at a 100 time lower concentration with CHO-derived rhCCL18 than with E. coli-derived rhCCL18. FT-ICR mass spectrometry of the intact chemokines showed that the rhCCL18 produced by CHO cells contained the 2 disulfide bonds Cys10-Cys34 and Cys11-Cys50, in clear contrast to the rhCCL18 derived from E. coli where the Cys10-Cys34 bond was absent. We found that reduction of the Cys10-Cys34 of the CHO-derived rhCCL18 resulted in a shift of its activity, reaching the same level as the E. coli-derived rhCCL18. These results demonstrate that the Cys10-Cys34 disulfide bond is involved in the function of CCL18.

Proteomics applied to the authentication of fish glue: application to a 17th century artwork sample.

This work provides the first identification of fish glue from a few micrograms of a 17(th) century artwork sample using an adapted proteomics approach. Fish glue has been widely used as a binder in various art objects such as paintings, manuscripts or polychrome objects however its authentication remains particularly challenging. The lack of information on fish species in genomic and proteomic databases represents a major drawback. A supplementary difficulty is provided by the historical sample features, i.e. a few micrograms of a 17(th) century polychrome object with a multilayered structure. SYPRO® Ruby staining was used as a screening technique to probe the presence of proteins in the sample cross-section. Results revealed the presence of several layers containing proteins among which a thin proteinaceous layer located between the silver leaf and the glaze. This thin layer is described as fish glue coating by historical sources but its composition has not been identified yet. The optimized methodology, based on high resolution mass spectrometry and adapted bioinformatic tools, was successfully applied to 50 µg of a polychromy sample and resulted in the identification of several collagen proteins. Extensive interpretation of data generated by tandem mass spectrometry allowed the identification of proteins from different biological origins. In particular, seven peptides specific to fish collagen proteins were identified for the first time proving the presence of fish glue in the sample and corroborating information found in historical texts dealing with the polychromy technique.

Towards analytically useful two-dimensional Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) achieves high resolution and mass accuracy, allowing the identification of the raw chemical formulae of ions in complex samples. Using ion isolation and fragmentation (MS/MS), we can obtain more structural information, but MS/MS is time- and sample-consuming because each ion must be isolated before fragmentation. In 1987, Pfändler et al. proposed an experiment for 2D FT-ICR MS in order to fragment ions without isolating them and to visualize the fragmentations of complex samples in a single 2D mass spectrum, like 2D NMR spectroscopy. Because of limitations of electronics and computers, few studies have been conducted with this technique. The improvement of modern computers and the use of digital electronics for FT-ICR hardware now make it possible to acquire 2D mass spectra over a broad mass range. The original experiments used in-cell collision-induced dissociation, which caused a loss of resolution. Gas-free fragmentation modes such as infrared multiphoton dissociation and electron capture dissociation allow one to measure high-resolution 2D mass spectra. Consequently, there is renewed interest to develop 2D FT-ICR MS into an efficient analytical method. Improvements introduced in 2D NMR spectroscopy can also be transposed to 2D FT-ICR MS. We describe the history of 2D FT-ICR MS, introduce recent improvements, and present analytical applications to map the fragmentation of peptides. Finally, we provide a glossary which defines a few keywords for the 2D FT-ICR MS field.