Development of a candidate reference measurement procedure by ID-LC-MS/MS for total tau protein measurement in cerebrospinal fluid (CSF).

OBJECTIVES: In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF). METHODS: To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode. RESULTS: A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high). CONCLUSIONS: The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.

Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment.

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.

Innate immunity probed by lipopolysaccharides affinity strategy and proteomics.

Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.

Harmonization and standardization of biofluid-based biomarker measurements for AT(N) classification in Alzheimer's disease.

Fluid biomarkers are currently measured in cerebrospinal fluid and blood for Alzheimer's disease diagnosis and are promising targets for drug development and for patients' follow-up in clinical trials. These biomarkers have been grouped in an unbiased research framework, the amyloid (Aß), tau, and neurodegeneration (AT[N]) biomarker system to aid patients' early diagnosis and stratification. Metrological approaches relying on mass spectrometry have been used for the development of reference materials and reference measurement procedures. Despite their excellent performances as clinical tools, fluid biomarkers often present an important between-laboratory variation. Standardization efforts were carried out on the biomarkers currently included in the AT(N) classification system, involving the collaboration of national metrology institutes, clinicians, researchers, and in vitro diagnostic providers. This article provides an overview of current activities towards standardization. These reference methods and reference materials may be used for recalibration of immunoassays and the establishment of standardized cutoff values allowing a better stratification of Alzheimer's disease patients. Highlights: The AT(N) biomarker system allows stratifying AD patients on the basis of biomarker profiles.Fluid biomarker measurements often present an important between-laboratory variation preventing the establishment of standardized cutoff values.Overview on the standardization initiatives involving the fluid biomarkers currently included in the AT(N) framework.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.

BACKGROUND: The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. RESULTS: Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis-Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 µM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. CONCLUSIONS: In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.

hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene.

By generating mRNA containing a premature termination codon (PTC), alternative splicing (AS) can quantitatively regulate the expression of genes that are degraded by nonsense-mediated mRNA decay (NMD). We previously demonstrated that AS-induced retention of part of intron 3 of rpL3 pre-mRNA produces an mRNA isoform that contains a PTC and is targeted for decay by NMD. We also demonstrated that overexpression of rpL3 downregulates canonical splicing and upregulates the alternative splicing of its pre-mRNA. We are currently investigating the molecular mechanism underlying rpL3 autoregulation. Here we report that the heterogeneous nuclear ribonucleoprotein (hnRNP) H1 is a transacting factor able to interact in vitro and in vivo with rpL3 and with intron 3 of the rpL3 gene. We investigated the role played by hnRNP H1 in the regulation of splicing of rpL3 pre-mRNA by manipulating its expression level. Depletion of hnRNP H1 reduced the level of the PTC-containing mRNA isoform, whereas its overexpression favored the selection of the cryptic 3' splice site of intron 3. We also identified and characterized the cis-acting regulatory elements involved in hnRNP H1-mediated regulation of splicing. RNA electromobility shift assay demonstrated that hnRNP H1 specifically recognizes and binds directly to the intron 3 region that contains seven copies of G-rich elements. Site-directed mutagenesis analysis and in vivo studies showed that the G3 and G6 elements are required for hnRNP H1-mediated regulation of rpL3 pre-mRNA splicing. We propose a working model in which rpL3 recruits hnRNP H1 and, through cooperation with other splicing factors, promotes selection of the alternative splice site.

Proteomic analysis of cells exposed to prefibrillar aggregates of HypF-N.

Several human diseases are associated with the deposition of stable ordered protein aggregates known as amyloid fibrils. In addition, a large wealth of data shows that proteins not involved in amyloidoses, are able to form, in vitro, amyloid-like prefibrillar and fibrillar assemblies indistinguishable from those grown from proteins associated with disease. Previous studies showed that early prefibrillar aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) are cytotoxic, inducing early mitochondria membrane depolarization, activation of caspase 9 and eventually cell death. To gain knowledge on the molecular basis of HypF-N aggregate cytotoxicity, we performed a differential proteomic analysis of NIH-3T3 cells exposed to HypF-N prefibrillar aggregates in comparison with control cells. Two-dimensional gel electrophoresis followed by protein identification by MALDI-TOF MS, allowed us to identify 21 proteins differentially expressed. The changes of the expression level of proteins involved in stress response (Hsp60 and 78 kDa glucose-regulated protein) and in signal transduction (Focal adhesion kinase1) appear particularly interesting as possible determinants of the cell fate. The levels of some of the differently expressed proteins were modified also in similar studies carried out on cells exposed to Abeta or alpha-synuclein aggregates, supporting the existence of shared features of amyloid cytotoxicity.

Glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights.

Bottom up proteomics requires efficient and selective pre-fractionation procedures to simplify the analysis of the enormous number of peptides resulting from the hydrolysis of a cellular extract enabling the detection, identification and the structural characterization of the post-translational modifications. Glycosylation, a well-known post-translational modification, plays a key role in the enormous complexity, and heterogeneity of the human blood serum proteome. Thereby, characterization of glycosylation from serum is a challenging task, even for the existing sophisticated analytical methodologies. Here we report a glycoproteomics study on the identification of even low abundant glycoproteins, including the localization of N-glycosylation sites and the glycan profiling in human sera from healthy and myocarditis affected donors. The strategy is simply based on proteolytic digestion of total serum proteins followed by a single enrichment step of glycopeptides on ConA lectin affinity chromatography. Glycopeptides were then deglycosylated by PNGaseF treatment and nano-liquid chromatography-electrospray ionization tandem mass spectrometry analyses of the free peptides provided the basis for both identification of the individual proteins and elucidation of their modification sites. Moreover, glycan profilings could be obtained by matrix-assisted laser desorption/ionization mass spectrometry analysis of the released oligosaccharides. Our data led to the identification of 68 different glycosylation sites within 49 different proteins. Moreover, the analyses carried out on glycans represent the first picture of a glycosylation pattern in myocardial lesions. As a whole, several differences in the glycosylation patterns from different sera were observed, thus indicating glycan profiling as a possible tool to discriminate among different diseases.

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein.

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway.

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated ß-Turn Structures to be Used as Probes in Autoimmune Diseases.

Synthetic sugar-modified peptides were identified as antigenic probes in the context of autoimmune diseases. The aim of this work is to provide a mechanistic study on the fragmentation of different glycosylated analogs of a synthetic antigenic probe able to detect antibodies in a subpopulation of multiple sclerosis patients. In particular the N-glucosylated type I' ß-turn peptide structure called CSF114(Glc) was used as a model to find signature fragmentations exploring the potential of multi-stage mass spectrometry by MALDI-LTQ Orbitrap. Here we compare the fragmentation of the glucosylated form of the synthetic peptide CSF114(Glc), bearing a glucose moiety on an asparagine residue, with less or non- immunoreactive forms, bearing different sugar-modifications, such as CSF114(GlcNAc), modified with a residue of N-acetylglucosamine, and CSF114[Lys(7)(1-deoxyfructopyranosyl)], this last one modified with a 1-deoxyfructopyranosyl moiety on a lysine at position 7. The analysis was set up using a synthetic compound specifically deuterated on the C-1 to compare its fragmentation with the fragmentation of the undeuterated form, and thus ascertain with confidence the presence on an Asn(Glc) within a peptide sequence. At the end of the study, our analysis led to the identification of signature neutral losses inside the sugar moieties to characterize the different types of glycosylation/glycation. The interest of this study lies in the possibility of applyimg this approach to the discovery of biomarkers and in the diagnosis of autoimmune diseases. Graphical Abstract .

Industrial waste based compost as a source of novel cellulolytic strains and enzymes.

Ninety bacteria isolated from raw composting materials were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. The bacteria producing the highest cellulolytic activity levels were identified by 16S rRNA sequencing as Bacillus licheniformis strain 1, Bacillus subtilis subsp. subtilis strain B7B, Bacillus subtilis subsp. spizizenii strain 6, and Bacillus amyloliquefaciens strain B31C. Cellulase activity production by the most productive strain B. amyloliquefaciens B31C was optimized in liquid culture varying the carbon source. Comparison of growth curves of B. amyloliquefaciens B31C at temperatures from 28 to 47 °C indicated its thermotolerant nature. Moreover, analysis of time courses of cellulase activity production in this thermal range showed that increase of temperature from 28 to 37 °C causes an increase of cellulase activity levels. Investigating the enzymes responsible for cellulase activity produced by B. amyloliquefaciens B31C by proteomic analyses, an endoglucanase was identified. It was shown that the purified enzyme catalyzes carboxymethylcellulose's hydrolysis following Michaelis-Menten kinetics with a K(M) of 9.95 mg ml(-1) and a v(max) of 284 µM min(-1) . It shows a retention of 90% of its activity for at least 144 h of incubation at 40 °C and exhibits a range of optimum temperatures from 50 to 70 °C.

Apolipoprotein A-I amyloidogenic variant L174S, expressed and isolated from stably transfected mammalian cells, is associated with fatty acids.

Sixteen variants of apolipoprotein A-I (ApoA-I) are associated with hereditary systemic amyloidoses, characterized by amyloid deposition in peripheral organs of patients. As these are heterozygous for the amyloidogenic variants, their isolation from plasma is impracticable and recombinant expression systems are needed. Here we report the expression of recombinant ApoA-I amyloidogenic variant Leu174 with Ser (L174S) in stably transfected Chinese hamster ovary-K1 cells. ApoA-I variant L174S was found to be efficiently secreted in the culture medium, from which it was isolated following a one-step purification procedure. Mass spectrometry analyses allowed the qualitative and quantitative definition of the amyloidogenic variant lipid content, which was found to consist of two saturated and two monounsaturated fatty acids. Interestingly, the same lipid species were found to be associated with the wild-type ApoA-I, expressed and isolated using the same cell system, with lower values of the lipid to protein molar ratios with respect to the amyloidogenic variant. A possible role of fatty acids in trafficking and secretion of apolipoproteins may be hypothesized.

Characterization of laccase isoforms produced by Pleurotus ostreatus in solid state fermentation of sugarcane bagasse.

Laccases are oxidative enzymes linked to biological degradation of lignin. The aim of this work was to evaluate the effect of inducers and different concentrations of nitrogen on production level of total laccase activity and pattern of laccase isoforms, produced in solid state fermentation of sugarcane bagasse by a selected strain of Pleurotus ostreatus. The addition of yeast extract 5 g/L, copper sulfate 150 µM and ferulic acid 2 mM provided highest enzymatic activity (167 U/g) and zymograms indicated the presence of six laccase isoforms (POXA1b, POXA3, POXC and three other isoforms). Results of protein identification by mass spectrometry confirmed the presence of POXC and POXA3 as the main isoenzymes, and also identified a glyoxal oxidase and three galactose oxidases. The fact that the isoenzyme POXA1b was not identified in the analyzed samples can be possibly explained by its sensitivity to protease degradation.

Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice.

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Technical advances in proteomics mass spectrometry: identification of post-translational modifications.

The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs.