Amadori rearrangement products as potential biomarkers for inborn errors of amino-acid metabolism.

The identification of disease biomarkers plays a crucial role in developing diagnostic strategies for inborn errors of metabolism and understanding their pathophysiology. A primary metabolite that accumulates in the inborn error phenylketonuria is phenylalanine, however its levels do not always directly correlate with clinical outcomes. Here we combine infrared ion spectroscopy and NMR spectroscopy to identify the Phe-glucose Amadori rearrangement product as a biomarker for phenylketonuria.

Confirmation of neurometabolic diagnoses using age-dependent cerebrospinal fluid metabolomic profiles.

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.

Next-generation metabolic screening: targeted and untargeted metabolomics for the diagnosis of inborn errors of metabolism in individual patients.

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.

NANS-mediated synthesis of sialic acid is required for brain and skeletal development.

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.

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis.

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan 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 (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background.

Autoreactivity against induced or upregulated abundant self-peptides in HLA-A*0201 following measles virus infection.

Infectious agents have been implied as causative environmental factors in the development of autoimmunity. However, the exact nature of their involvement remains unknown. We describe a possible mechanism for the activation of autoreactive T cells induced by measles virus (MV) infection.

Dynamics of measles virus protein expression are reflected in the MHC class I epitope display.

Following measles virus (MV) infection, viral peptides are presented to CTL by MHC class I molecules on infected antigen presenting cells at widely different epitope densities. Whereas three MV epitopes (MV-M(211-219), MV-F(438-446) and MV-H(30-38)) derived from different structural proteins occur at regular densities, one peptide derived from the non-structural C protein (MV-C(84-92)) fully dominates the MV peptide display in HLA class I molecules on end-stage-infected human B cells. Here we demonstrate that this hierarchy in MV epitope density is not a constant, but varies with progression of infection.

A measles virus glycoprotein-derived human CTL epitope is abundantly presented via the proteasomal-dependent MHC class I processing pathway.

Peptides derived from measles virus (MV) are presented by MHC class I molecules at widely divergent levels, but it is currently unknown how functional these levels are. Here, for the first time, we studied the natural occurrence and the underlying processing events of a known MV CTL epitope derived from the fusion glycoprotein (MV-F) and restricted via HLA-B*2705. Using MHC-peptide elution of MV-infected cells followed by sensitive mass spectrometry we determined the naturally occurring sequence to be RRYPDAVYL, corresponding to MV-F(438-446).

Comparison of various liquid chromatographic methods involving UV and atmospheric pressure chemical ionization mass spectrometric detection for the efficient trace analysis of phenylurea herbicides in various types of water samples.

The performance of mass spectrometric (MS) detection and UV detection in combination with reversed-phase liquid chromatography without and with the use of coupled column RPLC (LC-LC) has been compared for the trace analysis of phenylurea herbicides in environmental waters. The selected samples of this comparative study originated from an inter-laboratory study. For both detection modes, a 50 mm x 4.

A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

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 (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces.

A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.

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.

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry.

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.

Conformation and quantification of chloramphenicol in cow's urine, muscle and eggs by electron capture negative ion chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of residues of the antibiotic chloramphenicol in biological samples. The method is based on gas chromatography/negative ion chemical ionization mass spectrometry and uses (37Cl2)chloramphenicol as internal standard. Selective ion monitoring of four analyte-specific ions enables the determination of chloramphenicol levels in urine of 3 micrograms l-1 with a coefficient of variation of 8%.

Multi-immunoaffinity chromatography: a simple and highly selective clean-up method for multi-anabolic residue analysis of meat.

A method for the detection of nortestosterone (NT) in bovine muscle at levels below 1 microgram/kg is described, based on enzymatic digestion of the sample, clean-up by immunoaffinity chromatography after defatting and detection by gas chromatography-mass spectrometry (selected-ion monitoring). The immunoaffinity matrix was prepared after combining the isolated immunoglobulin G fractions from a rabbit antiserum raised against NT and methyltestosterone (MT). Its capacity per millilitre of gel was approximately 10 ng for each of the two steroids.