High-throughput nucleoside phosphate monitoring in mammalian cell fed-batch cultivation using quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Current methods for monitoring multiple intracellular metabolite levels in parallel are limited in sample throughput capabilities and analyte selectivity. This article presents a novel high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) for monitoring intracellular metabolite levels in fed-batch processes. The MALDI-TOF-MS method presented here is based on a new microarray sample target and allows the detection of nucleoside phosphates and various other metabolites using stable isotope labeled internal standards.

Homocysteine, cysteine, folate and vitamin B₁₂ status in type 2 diabetic patients with chronic kidney disease.

Background: Hyperhomocysteinemia (hHcy) is a risk factor in the progression of chronic kidney disease (CKD). In type 2 diabetes (T2D), hHcy is strongly associated with increased risk of cardiovascular disease. Vitamin B12 and folic acid supplementation have been reported to lower homocysteine (tHcy) levels, but no data on plasma tHcy, cysteine (Cys), folate and vitamin B12 levels in T2D-CKD patients are reported.

Fingerprinting breast cancer vs. normal mammary cells by mass spectrometric analysis of volatiles.

There is increasing interest in the development of noninvasive diagnostic methods for early cancer detection, to improve the survival rate and quality of life of cancer patients. Identification of volatile metabolic compounds may provide an approach for noninvasive early diagnosis of malignant diseases. Here we analyzed the volatile metabolic signature of human breast cancer cell lines versus normal human mammary cells.

Mass spectrometry research at the Laboratory for Organic Chemistry, ETH Zurich.

This contribution covers the most important activities of the Zenobi research group at the Organic Chemistry Laboratory, ETH Zurich. We work in a number of interrelated areas that encompass fundamental/mechanistic research, instrument and methods development, and applications. This is illustrated with examples from the mass spectrometric study of noncovalent interactions, using both native ESI and MALDI for ionization, the investigation of the gas-phase conformation of ionized bio-macromolecules, the use of ambient mass spectrometry for rapid, on-line analyses of, for example, exhaled breath, and the use of MALDI and microarray technologies for studying metabolites with extreme sensitivity, sufficient to probe the metabolites from single cells.

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance.

Quantification of saquinavir from lysates of peripheral blood mononuclear cells using microarrays and standard MALDI-TOF-MS.

Drug monitoring is usually performed by liquid chromatography coupled with optical detection or electrospray ionization mass spectrometry. More recently, matrix-assisted laser desorption/ionization (MALDI) in combination with triple quadrupole or Fourier-transform (FT) mass analyzers has also been reported to allow accurate quantification. Here, we present a strategy that employs standard MALDI time-of-flight (TOF) mass spectrometry (MS) for the sensitive and accurate quantification of saquinavir from an extract of blood peripheral mononuclear cells.

The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.

Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli.

Fluorescence resonance energy transfer of gas-phase ions under ultra high vacuum and ambient conditions.

We report evidence for fluorescence resonance energy transfer (FRET) of gas-phase ions under ultra high vacuum conditions (10(-9) mbar) inside a mass spectrometer as well as under ambient conditions inside an electrospray plume. Two different FRET pairs based on carboxyrhodamine 6G (donor) and ATTO590 or Bodipy TR (acceptor) dyes were examined and their gas-phase optical properties were studied. Our measurements indicate a different behavior for the two FRET pairs, which can be attributed to their different conformations in the gas phase.

Performances of capillary electrophoresis and HPLC methods in HbA1c determination: diagnostic accuracy in HbS and HbD-Iran variants' presence.

Background: Glycated hemoglobin (HbA1c) provides a useful estimate of mean glycemia in patients with diabetes and is directly related to risks for diabetes complications. The aim of this study is to compare a capillary electrophoresis method and two high-performance liquid chromatography (HPLC) cation-exchange analyzers (Variant II (Bio-Rad Laboratories, Inc., Hercules, CA) and G8 (Tosoh Biosciences, San Francisco, CA)) to identify the most reliable method in Hb variants' presence.

High-mass MALDI-MS using ion conversion dynode detectors: influence of the conversion voltage on sensitivity and spectral quality.

With the development of special ion conversion dynode (ICD) detectors for high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the mass-to-charge ratio is no longer a limiting factor. Although these detectors have been successfully used in the past, there is lack of understanding of the basic processes in the detector. We present a systematic study to investigate the performance of such an ICD detector and separate the contributions of the MALDI process from the ones of the ion-to-secondary ion and the secondary ion-to-electron conversions.

Real-time breath analysis with active capillary plasma ionization-ambient mass spectrometry.

On-line analysis of exhaled human breath is a growing area in analytical science, for applications such as fast and non-invasive medical diagnosis and monitoring. In this work, we present a novel approach based on ambient ionization of compounds in breath and subsequent real-time mass spectrometric analysis. We introduce a plasma ionization source for this purpose, which has no need for additional gases, is very small, and is easily interfaced with virtually any commercial atmospheric pressure ionization mass spectrometer (API-MS) without major modifications.

Synthesis of two-dimensional analogues of copolymers by site-to-site transmetalation of organometallic monolayer sheets.

Monolayer sheets have gained attention due to the unique properties derived from their two-dimensional structure. One of the key challenges in sheet modification/synthesis is to exchange integral parts while keeping them intact. We describe site-to-site transmetalation of Zn(2+) in the netpoints of cm(2)-sized, metal-organic sheets by Fe(2+), Co(2+), and Pb(2+).

Influence of dialysis techniques and alternate vitamin supplementation on homocysteine levels in patients with known MTHFR genotypes.

Background: Treatment with folic acid and vitamin B12 appears capable of reducing total plasma homocysteine levels (tHcy), but it is unknown whether vitamin B12 alone reduces tHcy values. In this study we investigate the effects of alternate vitamins supplementation on homocysteine levels in patients treated by diffusive and convective dialysis techniques.

Methods: 74 patients were randomized blindly into two groups of 37 subjects each.

Breath analysis in real time by mass spectrometry in chronic obstructive pulmonary disease.

Background: It has been suggested that exhaled breath contains relevant information on health status.

Objectives: We hypothesized that a novel mass spectrometry (MS) technique to analyze breath in real time could be useful to differentiate breathprints from chronic obstructive pulmonary disease (COPD) patients and controls (smokers and nonsmokers).

Methods: We studied 61 participants including 25 COPD patients [Global Initiative for Obstructive Lung Disease (GOLD) stages I-IV], 25 nonsmoking controls and 11 smoking controls.

Single-cell metabolomics: analytical and biological perspectives.

There is currently much interest in broad molecular profiling of single cells; a cell's metabolome-its full complement of small-molecule metabolites-is a direct indicator of phenotypic diversity of single cells and a nearly immediate readout of how cells react to environmental influences. However, the metabolome is very difficult to measure at the single-cell level because of rapid metabolic dynamics, the structural diversity of the molecules, and the inability to amplify or tag small-molecule metabolites. Measurement techniques including mass spectrometry, capillary electrophoresis, and, to a lesser extent, optical spectroscopy and fluorescence detection have led to impressive advances in single-cell metabolomics.

Human endogenous retroviruses and ADHD.

Objectives: Several lines of evidences suggest that human endogenous retroviruses (HERVs) are implicated in the development of many complex diseases with a multifactorial aetiology and a strong heritability, such as neurological and psychiatric diseases. Attention deficit hyperactivity Disorder (ADHD) is a neurodevelopmental disorder that results from a complex interaction of environmental, biological and genetic factors. Our aim was to analyse the expression levels of three HERV families (HERV-H, K and W) in patients with ADHD.

A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear.

DNA oligonucleotides: a model system with tunable binding strength to study monomer-dimer equilibria with electrospray ionization-mass spectrometry.

Electrospray ionization (ESI) is increasingly used to measure binding strengths, but it is not always clear whether the ESI process introduces artifacts. Here we propose a model monomer-dimer equilibrium system based on DNA oligonucleotides to systematically explore biomolecular self-association with the ESI-mass spectrometry (MS) titration method. The oligonucleotides are designed to be self-complementary and have the same chemical composition and mass, allowing for equal ionization probability, ion transmission, and detection efficiency in ESI-MS.

Real-time monitoring of exhaled drugs by mass spectrometry.

Future individualized patient treatment will need tools to monitor the dose and effects of administrated drugs. Mass spectrometry may become the method of choice to monitor drugs in real time by analyzing exhaled breath. This review describes the monitoring of exhaled drugs in real time by mass spectrometry.

Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: role of in-source fragmentation.

Nucleotides are key players in the central energy metabolism of cells. Here we show how to estimate the energy charge from cell lysates by direct negative ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using 9-aminoacridine as matrix. We found a high level of in-source decay of all the phosphorylated nucleotides, with some of them producing considerable amounts of adenosine-5'-diphosphate (ADP) fragment ions.

Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS.

Atherosclerosis, dyslipidemia, and inflammation: the significant role of polyunsaturated Fatty acids.

Phospholipids play an essential role in cell membrane structure and function. The length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity, transport systems, activity of membrane-bound enzymes, and susceptibility to lipid peroxidation. The fatty acid profile of serum lipids, especially the phospholipids, reflects the fatty acid composition of cell membranes.

Analysis of single algal cells by combining mass spectrometry with Raman and fluorescence mapping.

In order to investigate metabolic properties of single cells of freshwater algae (Haematococcus pluvialis), we implement matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with microspectroscopic mapping. Straightforward coupling of these two detection platforms was possible thanks to the self-aliquoting properties of micro-arrays for mass spectrometry (MAMS). Following Raman and fluorescence imaging, the isolated cells were covered with a MALDI matrix for targeted metabolic analysis by MALDI-MS.

Analysis of the exhalome: a diagnostic tool of the future.

Investigations on breath analysis have provided preliminary data on its potential in the noninvasive diagnosis of lung diseases. Although the conventional comparisons of exhaled breath in study populations (ie, diseased vs healthy) may help to identify patients with various lung diseases, we believe that the analysis of exhaled breath holds promise beyond this scenario. On the basis of preliminary findings, we hypothesize that breath analysis (1) could be applied not only to identify patients with lung disease but also to better phenotype healthy subjects at risk and patients with a particular disease, which is in-line with current efforts toward individualized medicine; (2) could be useful in estimating internal body time to determine the optimal time of drug administration, thereby maximizing drug activity and reducing toxicity (chronopharmacology); and (3) could be applied to monitor drugs or drug metabolites, thus, enhancing adherence to prescribed medications and enabling studies on pharmacokinetics.

Self-aliquoting microarray plates for accurate quantitative matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors.