The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed “next-generation metabolic screening” (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the “various forms of chromatography mass spectrometry (XCMS)” software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term “open the metabolome” analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs.Electronic supplementary materialThe online version of this article (10.1007/s10545-017-0131-6) contains supplementary material, which is available to authorized users.
We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition. DOI: https://doi.org/10. 1038/ng.3578 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-130493 Accepted Version Originally published at: van Karnebeek, Clara D M; Bonafé, Luisa; Wen, Xiao-Yan; Tarailo-Graovac, Maja; Balzano, Sara; RoyerBertrand, Beryl; Ashikov, Angel; Garavelli, Livia; Mammi, Isabella; Turolla, Licia; Breen, Catherine; Donnai, Dian; Cormier, Valerie; Heron, Delphine; Nishimura, Gen; Uchikawa, Shinichi; Campos-Xavier, Belinda; Rossi, Antonio; Hennet, Thierry; Brand-Arzamendi, Koroboshka; Rozmus, Jacob; Harshman, Keith; Stevenson, Brian J; Girardi, Enrico; Superti-Furga, Giulio; Dewan, Tammie; Collingridge, Alissa; Halparin, Jessie; Ross, Colin J; Van Allen, Margot I;et al (2016). NANS-mediated synthesis of sialic acid is required for brain and skeletal development. Nature Genetics, 48 (7) insights into the molecular basis of neurocognitive impairment allows for the development and 89 application of targeted therapeutic strategies 5 . Although less frequent than IDD, genetic disorders 90 affecting skeletal development and growth (commonly called the "skeletal dysplasias") are a 91 group of over 500 distinct disorders 6 . Studying their molecular basis has provided precious 92 insights into the many factors necessary for skeletal development, ranging from minerals and 93 structural molecules to enzymes, to signaling molecules and transcription factors 6,7 . We report 94here a genetic disorder presenting with a combination of severe IDD with skeletal dysplasia and 95 short stature. Our data show that its pathogenic basis is an inborn error of metabolism that 96 affects the endogenous synthesis of N-acetyl neuraminic acid (NeuNAc; sialic acid). Exploration 97 of the biochemical and molecular features of this disorder provides new information on the role 98 of sialic acid in the development of brain and bone. 99 100 RESULTS 101 Clinical and radiographic phenotype of N...
We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA‐A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection‐induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization / mass spectrometry (μLC‐ESI / MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA‐peptide complexes, but had similar binding capacities to HLA‐A*0201. The most abundant viral peptide species, identified as residues 84 – 92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 105 copies per cell) and was immunogenic in HLA‐A2 / Kb‐transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA‐A*0201 binding capacity. Thus, here we report for the first time the direct discovery through μLC‐ESI / MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.
A microcapillary column switching high-performance liquid chromatography (HPLC) system was developed for the separation of major histocompatibility complex (MHC) class I associated peptides. Combination of the column switching system with electrospray ionization mass spectrometry (ESIMS) enabled the detection and identification of the peptides at low-femtomole levels. Sample volumes of 30-50 microL were injected and concentrated onto a short, 100-micron-i.d. precolumn. The precolumn was coupled to a 100-micron-i.d. reversed-phase analytical HPLC column via a six-port valve. Peptides were separated on the analytical column using an ESI-compatible mobile phase at a flow rate of 0.5 microL/min. Peptides were eluted directly into the ESI source of either a magnetic sector MS or an ion trap MS. Peptides associated with human leukocyte antigen A*0201 molecules were determined in immunoaffinity-purified extracts from either measles virus infected cells or uninfected cells by microcapillary column switching HPLC-ESIMS. The approach toward detection of virus-specific peptides we used was based on the comparison of ion chromatograms obtained from the LC-MS analysis of extracts from virally infected cells and their uninfected counterparts. In this way, the molecular mass of peptides unique to virus infected cells was obtained. The utility of the system is demonstrated by the identification of a candidate epitope. Microcapillary column switching HPLC was used along with ESI ion trap tandem MS to identify the naturally processed viral peptide KLWESPQEI. This peptide was found to derive from the measles virus nonstructural protein C. The approach described here provides a versatile and sensitive method for the direct identification of viral peptides associated with MHC class I molecules.
The applicability of ultra-performance liquid chromatography (UPLC) combined with fullscan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (Ͻ3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further. (J Am Soc Mass Spectrom 2009, 20, 451-463)
Timely diagnosis is essential for patients with neurometabolic disorders to enable targeted treatment. Next‐Generation Metabolic Screening (NGMS) allows for simultaneous screening of multiple diseases and yields a holistic view of disturbed metabolic pathways. We applied this technique to define a cerebrospinal fluid (CSF) reference metabolome and validated our approach with patients with known neurometabolic disorders. Samples were measured using ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry followed by (un)targeted analysis. For the reference metabolome, CSF samples from patients with normal general chemistry results and no neurometabolic diagnosis were selected and grouped based on sex and age (0‐2/2‐5/5‐10/10‐15 years). We checked the levels of known biomarkers in CSF from seven patients with five different neurometabolic disorders to confirm the suitability of our method for diagnosis. Untargeted analysis of 87 control CSF samples yielded 8036 features for semiquantitative analysis. No sex differences were found, but 1782 features (22%) were different between age groups (q < 0.05). We identified 206 diagnostic metabolites in targeted analysis. In a subset of 20 high‐intensity metabolites and 10 biomarkers, 17 (57%) were age‐dependent. For each neurometabolic patient, ≥1 specific biomarker(s) could be identified in CSF, thus confirming the diagnosis. In two cases, age‐matching was essential for correct interpretation of the metabolomic profile. In conclusion, NGMS in CSF is a powerful tool in defining a diagnosis for neurometabolic disorders. Using our database with many (age‐dependent) features in CSF, our untargeted approach will facilitate biomarker discovery and further understanding of mechanisms of neurometabolic disorders.
An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.
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