Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.

Investigation of space charge effects and ion trapping capacity on direct introduction ultra-high-resolution mass spectrometry workflows for metabolomics.

Ultra-high-resolution mass spectrometry, in the absence of chromatography, is finding its place for direct analyses of highly complex mixtures, such as those encountered during untargeted metabolomics screening. Advances, however, have been tempered by difficulties such as uneven signal suppression experienced during electrospray ionization. Moreover, ultra-high-resolution mass spectrometers that use Orbitrap and ICR analyzers both suffer from limited ion trapping capacities, owing principally to space-charge effects. This study has evaluated and contrasted the above two types of Fourier transform mass spectrometers for their abilities to detect and identify by accurate mass measurement, small molecule metabolites present in complex mixtures. For these direct introduction studies, the Orbitrap Fusion showed a major advantage in terms of speed of analysis, enabling detection of 218 of 440 molecules (<2 ppm error, 500 000 resolution at m/z 200) present in a complex mixture in 5 min. This approach is the most viable for high-throughput workflows, such as those used in investigations involving very large cohorts of metabolomics samples. From the same mixture, 183 unique molecules were observed by FT-ICR in the broadband mode, but this number was raised to 235 when "selected ion monitoring-stitching" (SIM-stitching) was employed (<0.1 ppm error, 7 T magnet with dynamic harmonization cell, 1.8 million resolution at m/z 200, both cases). SIM-stitching FT-ICR thus offered the most complete detection, which may be of paramount importance in situations where it is essential to obtain the most complete metabolic profile possible. This added completeness, however, came at the cost of a more lengthy analysis time (120 min including manual treatment). Compared to the data presented here, future automation of processing, plus the use of absorption mode detection, segmented ion detection (stepwise detection of smaller width m/z sections), and higher magnetic field strengths, can substantially reduce FT-ICR acquisition times.

Suspect and non-targeted screening of chemicals of emerging concern for human biomonitoring, environmental health studies and support to risk assessment: From promises to challenges and harmonisation issues.

Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017-2021) are described, with an emphasis on harmonisation measures.

Comprehensive Preterm Breast Milk Metabotype Associated with Optimal Infant Early Growth Pattern.

Early nutrition impacts preterm infant early growth rate and brain development but can have long lasting effects as well. Although human milk is the gold standard for feeding new born full-term and preterm infants, little is known about the effects of its bioactive compounds on breastfed preterm infants' growth outcomes. This study aims to determine whether breast milk metabolome, glycome, lipidome, and free-amino acids profiles analyzed by liquid chromatography-mass spectrometry had any impact on the early growth pattern of preterm infants. The study population consisted of the top tercile-Z score change in their weight between birth and hospital discharge ("faster grow", n = 11) and lowest tercile ("slower grow", n = 15) from a cohort of 138 premature infants (27⁻34 weeks gestation). This holistic approach combined with stringent clustering or classification statistical methods aims to discriminate groups of milks phenotype and identify specific metabolites associated with early growth of preterm infants. Their predictive reliability as biomarkers of infant growth was assessed using multiple linear regression and taking into account confounding clinical factors. Breast-milk associated with fast growth contained more branched-chain and insulino-trophic amino acid, lacto-N-fucopentaose, choline, and hydroxybutyrate, pointing to the critical role of energy utilization, protein synthesis, oxidative status, and gut epithelial cell maturity in prematurity.

Mechanistic study of competitive releases of H2O, NH3 and CO2 from deprotonated aspartic and glutamic acids: Role of conformation.

The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3 experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways. These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2 as observed in the MS3 experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D-H)-NH3]-during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.

MS-based approaches to unravel the molecular complexity of proprotein-derived biomarkers and support their quantification: the examples of B-type natriuretic peptide and apelin peptides.

The specific forms of described protein biomarkers that occur in human blood are not yet fully established. Even though B-type natriuretic peptide (BNP) and N-terminal proBNP are now well known markers of heart failure and other cardiac disorders, several studies yielded highly controversial results reporting various truncated, multimerized or modified forms in human blood. Similar discrepancies were observed for other biomarkers also originating from proproteins, such as the apelin peptides. The drawback of most of these studies is that they used methods with low resolving power, such as immunoassays after HPLC separation. MS-based techniques may be able to avoid such flaws. In this review, we discuss the usefulness of MS-based approaches for the characterization of circulating forms of peptide biomarkers that originate from a given proprotein. Two particular examples are discussed in detail: BNP-related peptides and some more putative biomarkers of heart failure, the apelin peptides.

Identification and characterization of apelin peptides in bovine colostrum and milk by liquid chromatography-mass spectrometry.

Apelin peptides were recently identified as endogenous ligands of the APJ receptor. It has been hypothesized that these peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development. As apelin peptides may have different effects on the APJ receptor as a function of their size, knowledge of their exact structure in early milk is essential to clarify their action in gastrointestinal tract development. Bovine colostrum is thought to contain high concentrations of a wide diversity of apelin peptides, but none of them have yet been rigorously characterized. To identify and monitor apelin peptides in bovine colostrum, we developed a cation exchange extraction step followed by untargeted liquid chromatography coupled to high resolution and high mass accuracy mass spectrometry (LTQ-Orbitrap). Using this approach, we characterized 46 endogenous apelin peptides in bovine colostrum, which varied in relative abundance from one colostrum to another. Mature as well as commercial milk samples were also studied. Taken together, our data demonstrate that the multiplicity and variability of apelin peptides are biologically relevant and change during milk maturation to reach a more constant composition in mature milk.

Mass spectrometric investigation of molecular variability of grass pollen group 1 allergens.

Natural grass pollen allergens exhibit a wide variety of isoforms. Precise characterization of such microheterogeneity is essential to improve diagnosis and design appropriate immunotherapies. Moreover, standardization of allergen vaccine production is a prerequisite for product safety and efficiency. Both qualitative and quantitative analytical methods are thus required to monitor and control the huge natural variability of pollens, as well as final product quality. A proteomic approach has been set up to investigate in depth the structural variability of five group 1 allergens originating from distinct grass species (Ant o 1, Dac g 1, Lol p 1, Phl p 1, and Poa p 1). Whereas group 1 is the most conserved grass pollen allergen, great variations were shown between the various isoforms found in these five species using mass spectrometry, with many amino acid exchanges, as well as variations in proline hydroxylation level and in main N-glycan motifs. The presence of O-linked pentose residues was also demonstrated, with up to three consecutive units on the first hydroxyproline of Ant o 1. In addition, species-specific peptides were identified that might be used for product authentication or individual allergen quantification. Lastly, natural or process-induced modifications (deamidation, oxidation, glycation) were evidenced, which might constitute useful indicators of product degradation.

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII.

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.