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The nematode Haemonchus contortus (the barber's pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.

The nematode Haemonchus contortus (the barber’s pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.

The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.

The HIPPO pathway plays an important role in cancer progression and one of its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different family members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that TEADs are partially palmitoylated. Activity and stability of TEAD proteins are regulated by this modification. Here, we attempt to better understand the palmitoylation status of TEAD proteins in cells. In a first step, we have developed LC/MS/MS-based assays for the detection of palmitoylated TEAD peptides using recombinant TEAD1 protein. Subsequently, we have set out to analyze endogenous TEAD proteins using YAP1 activated malignant mesothelioma H2052 tumor cells. H2052 tumor cells express preferentially TEAD1 protein. TEAD1 was immunoprecipitated from H2052 tumor cells and subjected to peptidic digest and LC/MS/MS analysis. Peptide mapping permitted to confirm the expected site of palmitoylation (cysteine 359 of TEAD1). In addition, novel sites of both palmitoylation and myristoylation were detected on the lysine residue 336 of TEAD1. While these hydrophobic peptides could not be recovered after in-gel digestion of immunoprecipitation enriched TEAD1 of H2052 cells, digestion performed directly on immunoprecipitation beads allowed us to recover those peptides and hence to confirm the existence of TEAD lysine-myristoylation and lysine-palmitoylation in cells. In this study, we show the development of an MS-based strategy to characterize post translational modifications directly on endogenous TEAD1 proteins present in H2052 tumor cells. Citation Format: Armelle Buzy, Olivier Guichard, Jean-Luc Zacchayus, Olivier Venier, Olivier Courtin, Iris Valtingojer, Laurent Debussche, Vincent Mikol, Jean-Claude Guillemot. In vitro and in cellulo mass spectrometry study of TEAD1 palmitoylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4868.

Electrospray ionization mass spectrometry is shown to offer excellent characterization of linear oligomeric polysulfides H(SC2H4OCH2OC2H4S)nH with n = 1–24. Analysis of the mass spectra reveals the presence of individual oligomers, the presence in certain oligomers of repeat units containing additional oxyalkylene groups (and in some cases a monosulfide link rather than disulfide) and the presence of end groups such as epoxy. The attachment of polyacrylates during cure can also be detected. Assignments of individual peaks can be confirmed by collision-induced decomposition and precursor ion experiments: the fragmentation pathways are relatively few except for linear polysulfides of complex structure. The best signal-to-noise conditions are achieved with low-to-medium cone voltages and a mobile phase of acetone containing 0.5% KI.

Electrospray ionization mass spectrometry provides a highly detailed picture of the various species present in the high energy material poly(3-nitratomethyl-3-methyloxetane), also known as polynimmo. Polynimmo contains up to 18 cyclic oligomers in addition to the dominant tetramer, the highest species detected containing 22 repeat units. Linear oligomers from the tetramer up to species of mass 3200 Da are detected, affording the characterization of several new combinations of end groups X, Y in X-[nimmo] n -Y. Various cations can be used to generate the cationized species, and highly specific cation cyclic oligomer interactions are apparent in the adducts with Na + , K + , NH 4 + and H + : some of these can be rationalized through molecular modelling calculations. The cone voltage applied in the e.s.i. experiment is highly significant in influencing the relative abundances of high- and low-molecular mass ions and in the relative amounts of H + and NH 4 + adducts in cationization experiments with NH 4 Cl. Despite giving highly accurate relative molar masses of individual species within polynimmo, the e.s.i. technique fails to give accurate molecular mass distributions, especially as no doubly- or triply-charged ions are found.

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.

Polysulfide oligomers H(SC 2 H 4 OCH 2 OC 2 H 4 S) n H with n = 1–24 can be characterized by electrospray ionization mass spectrometry. Samples prepared by the conventional method are shown to contain up to eight series of oligomers differing in structure by one of a small group of variants in one (or sometimes two) of the repeat units, e.g. with one less S atom or an additional CH 2 O group. The composition of individual ions can be confirmed by collision-induced decomposition of cationized polysulfides, which yields unambiguous spectra of the fragmented ions.

The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.

The thermal degradation of poly(propylene oxide) (PPO), M n = 2000, can be characterized by electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI spectra of partially degraded PPO provide strong support for the thermal degradation pathway previously suggested by Griffiths et al. and Lemaire et al. Although these pathways differ in detail, it is not possible to distinguish between them from the masses of the resultant degradation species. Gel permeation chromatography data indicate that both mass spectrometric methods emphasize the presence of low-mass material, particularly in the degraded samples. This is attributed to the different sensitivities of the two techniques and some in situ fragmentation during mass spectrometric analysis.

Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec/Elitek), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme contains a cysteine adduct on Cys(103). In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys(35) and Cys(290)) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.

The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phosphorylated at Ser374. Its molecular mass measured by mass spectrometry (71,890 +/- 7 Da) is about 30 Da higher than the mass calculated from the sequence data (71,860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid, Eurypelma californicum, and 399 with chain alpha of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a, d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.

N,N′-dicyclohexylcarbodiimide (DCCD) interacts with isolated ceroid lipofuscinosis protein (CLP), a model for subunit c. The products are (a) CLP–DCCD adduct (+206 Da), presumably by addition at Glu-58; (b) acetyl–CLP–DCCD (+42 Da + 206 Da); (c) acetyl–CLP (+42 Da). Under specific conditions, additional DCCD adducts are formed; CLP–DCCD–DCCD (+206 Da + 206 Da) and acetyl–CLP–DCCD–DCCD (+42 Da + 206 Da + 206 Da). Acetylation utilises the acetate present in CLP preparations as a buffer and the site is shown to be located within amino acids 1–9 (probably Lys-7 or N-terminal aspartate). Acetyl–CLP is shown to be the primary product but, in addition, acylation by other fatty acids (myristate, palmitate, oleate and stearate) can be demonstrated in low acetate concentrations. Oligomycin is shown to modify some interactions of CLP with DCCD but venturicidin has little or no effect.

The use of electrospray tandem mass spectrometry is demonstrated for the characterisation of the polypeptide chains of normal human haemoglobin and a number of β-chain variants. Rapid screening of aberrant haemoglobins is achieved by the analysis of diluted blood without need for pretreatment or sample work-up. Gas-phase sequencing of the intact polypeptide chains shows that almost half of the amino acid sequence may be realised by this approach. More comprehensive coverage of the sequence (in excess of 80%) is given by cyanogen bromide digestion of whole blood followed by gas-phase sequencing of the resulting peptide fragments. The simplicity of the cyanogen bromide peptide map, accounting for the entire α- and β-chain sequences, is a distinct advantage of the method. Limitations of characterising aberrant proteins in triple quadrupole instruments are discussed with respect to mass accuracy and the effect of fragment ion charge state on detecting amino acid substitutions.

ESI MS is found to an effective tool for the analysis of poly(methyl methacrylate); optimum conditions involve the use of K+ counter ion at relatively high cone voltage; ESI MS is used for end-group and copolymer compositional analysis.

Electrospray ionisation (ESI) has been employed in conjunction with tandem mass spectrometry (MS/MS) on a tandem quadrupole mass spectrometer for the analysis of five common organic polymer additiv...

The loss of 60 u from protonated peptide ions containing an arginine residue at the C-terminus has been investigated by means of low energy tandem mass spectrometry. The lowest energy conformation of singly charged bradykinin is thought to involve a salt-bridge structure, which may lead to the formation of two isomeric forms. It is thought that one isomer retains the ionizing proton at the C-terminal end of the peptide, leading to the formation of the [bn−1 + H + OH]+ fragment ion, and the other isomer retains the charge at the N-terminus, leading to the formation of the [M + H − 60]+ fragment ion. It was found that the formation of the [M + H − 60]+ ion occurs only from singly charged precursor ions. In addition, the loss of 60 u occurs from peptides in which the charge is localized at the N-terminus. These results indicate that the mechanism of formation of the [M + H − 60]+ ion may be driven by a charge-remote process.

Introduction Tau hyperphosphorylation and neurofibrillary tangles are histopathologic hallmarks of tauopathies. Histamine H3-receptor antagonists have been proposed to reduce tau hyperphosphorylation in preclinical models. Methods We evaluated the ability of SAR110894, a selective histamine H3-receptor antagonist, to inhibit tau pathology and prevent cognitive deficits in a tau transgenic mouse model (THY-Tau22). Results SAR110894 treatment for 6 months (but not 2 weeks) in THY-Tau22 mice decreased both tau hyperphosphorylation at pSer396-pSer404 (AD2 signal) in the hippocampus and the number of AT8 (pSer199/202-Thr205) positive cells in the cortex and decreased the formation of neurofibrillary tangles in the cortex, hippocampus, and amygdala. Macrophage inflammatory protein 1-alpha messenger RNA expression was decreased in the hippocampus. SAR110894 also prevented episodic memory deficits, and this effect was still detected after treatment washout. Discussion Long-term SAR110894 treatment could have potential disease modifying activity in neurodegenerative tauopathies.