Johann Far

Detection and identification of hydrophilic selenium compounds in selenium-rich yeast by size exclusion–microbore normal-phase HPLC with the on-line ICP–MS and electrospray Q-TOF-MS detection

Normal-phase HPLC and hydrophilic interaction HPLC (HILIC) were investigated for the separation of selenometabolites in a water extract of Se-rich yeast prior to their detection by ICP-MS and identification by electrospray MS/MS. The targeted fraction was a low-abundant fraction co-eluting with salt and sulfur analogues in size-exclusion chromatography which has so far been inaccessible to Se speciation studies. The optimization of the separation conditions resulted in the highest separation efficiency when HILIC was used and elution was carried out isocratically with a low concentration ammonium acetate buffer (1 mM ammonium acetate/10 mM acetic acid) in 80% acetonitrile. Out of 15 peaks observed with the Se-specific ICP-MS detection 12 was identified by electrospray Q-TOF MS/MS (2,3-dihydroxypropionyl (DHP)-Se-methylselenocysteine [M+H]+: 272, Se-methyl-gamma-glutamyl-selenocysteinylglycine dioxide [M+H]+: 402, gamma-glutamyl-Se-methylselenocysteine [M+H]+: 313; isomers of gamma-glutamylselenocystathionine [M+H]+: 400; Se-methyl-selenoglutathione [M+H]+: 370, isomers of N-acetylselenocystathionine [M+H]+: 313, 2,3-DHP-selenohomolanthionine [M+H]+: 373, isomers of 2,3-DHP-selenocystathionine [M+H]+: 359, 2,3-DHP-selenolanthionine [M+H]+: 345 and selenohomolanthionine [M+H]+: 285).

Identification of anionic selenium species in Se-rich yeast by electrospray QTOF MS/MS and hybrid linear ion trap/orbitrap MSn.

An analytical approach allowing the identification of unknown selenium metabolites in selenium-rich yeast was described. Anion-exchange HPLC of the Se-metabolome fraction co-eluting with salts in size-exclusion chromatography allowed the separation of nine selenium species (excluding isomers and selenate) as monitored by inductively coupled plasma mass spectrometry (ICP MS). The individual fractions were analyzed by electrospray QTOF MS/MS and hybrid linear ion trap/Orbitrap MSn after sample introduction by reversed-phase nanoHPLC and by hydrophilic interaction LC (HILIC), respectively. Out of the nine detected species, eight were identified on the basis of accurate mass measurements and collision induced dissociation/fragmentation information. Seven Se-species (selenohomolanthionine, γ-Glu-selenocystathionine, 2,3-DHP-selenocystathionine, N-acetyl-selenocystathionine, 2,3-DHP-selenohomolanthionine, Se-methyl-selenoglutathione, and 2,3-DHP-Se-methylselenocysteine) were reported for the first time in Se-rich yeast, five of them have never been reported in any biological sample before.

Interlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.

Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes.

Supramolecular influence on cis–trans isomerization probed by ion mobility spectrometry

We used tandem ion mobility spectrometry measurements to investigate how the photo-isomerization of a chromophore (a methylpyridinium derivative) is affected by the complexation with a crown ether. A dramatic blue-shift of the photo-isomerization spectrum was observed upon complexation, which could be well reproduced by ab initio calculations. Our results support that the observed changes in the photo-physical properties of the chromophore originate from the charge-solvation of its pyridinium moiety by the host cage.

Evaluation of a new low sheath–flow interface for CE-MS

The current trend for increasing technical complexity in the field of CE‐ESI‐MS interfaces has incited for more accessible alternatives. In this work, a simple low sheath‐flow ESI interface operating in the submicroliter nanospray regime without nebulizing gas assistance was evaluated. The use of sheath liquid enabled improving the ionization of the analytes, while the absence of nebulizing gas minimized sample dilution and loss of efficiency. After a rapid qualification, the effect of main operational parameters such as sheath liquid composition and flow rate, working distance and ESI potential was studied. Simulation of the mixing processes inside the Taylor cone proved its size to be of utmost importance in band broadening processes. As a proof of concept, the interface was eventually applied to a set of representative basic drugs analyzed by CE‐TOF/MS. Limits of detection reached the 25–100 ppb range with suitable robustness and repeatability results. This design has demonstrated good performance while being simple and accessible to the user.

Multiple Gas-Phase Conformations of a Synthetic Linear Poly(acrylamide) Polymer Observed Using Ion Mobility-Mass Spectrometry

AbstractIon mobility-mass spectrometry (IM-MS) has emerged as a powerful separation and identification tool to characterize synthetic polymer mixtures and topologies (linear, cyclic, star-shaped,…). Electrospray coupled to IM-MS already revealed the coexistence of several charge state-dependent conformations for a single charge state of biomolecules with strong intramolecular interactions, even when limited resolving power IM-MS instruments were used. For synthetic polymers, the sample’s polydispersity allows the observation of several chain lengths. A unique collision cross-section (CCS) trend is usually observed when increasing the degree of polymerization (DP) at constant charge state, allowing the deciphering of different polymer topologies. In this paper, we report multiple coexisting CCS trends when increasing the DP at constant charge state for linear poly(acrylamide) PAAm in the gas phase. This is similar to observations on peptides and proteins. Biomolecules show in addition population changes when collisionally heating the ions. In the case of synthetic PAAm, fragmentation occurred before reaching the energy for conformation conversion. These observations, which were made on two different IM-MS instruments (SYNAPT G2 HDMS and high resolution multi-pass cyclic T-Wave prototype from Waters), limit the use of ion mobility for synthetic polymer topology interpretations to polymers where unique CCS values are observed for each DP at constant charge state. Graphical Abstractᅟ

Comprehensive Ion Mobility Calibration: Poly(ethylene oxide) Polymer Calibrants and General Strategies

Ion mobility (IM) is now a well-established and fast analytical technique. The IM hardware is constantly being improved, especially in terms of the resolving power. The Drift Tube (DTIMS), the Traveling Wave (TWIMS), and the Trapped Ion Mobility Spectrometry (TIMS) coupled to mass spectrometry are used to determine the Collision Cross-Sections (CCS) of ions. In analytical chemistry, the CCS is approached as a descriptor for ion identification and it is also used in physical chemistry for 3D structure elucidation with computational chemistry support. The CCS is a physical descriptor extracted from the reduced mobility (K0) measurements obtainable only from the DTIMS. TWIMS and TIMS routinely require a calibration procedure to convert measured physical quantities (drift time for TWIMS and elution voltage for TIMS) into CCS values. This calibration is a critical step to allow interinstrument comparisons. The previous calibrating substances lead to large prediction bands and introduced rather large uncertainties during the CCS determination. In this paper, we introduce a new IM calibrant (CCS and K0) using singly charged sodium adducts of poly(ethylene oxide) monomethyl ether (CH3O-PEO-H) for positive ionization in both helium and nitrogen as drift gas. These singly charged calibrating ions make it possible to determine the CCS/K0 of ions having higher charge states. The fitted calibration plots exhibit larger coverage with less data scattering and significantly improved prediction bands and uncertainties. The reasons for the improved CCS/K0 accuracy, advantages, and limitations of the calibration procedures are also discussed. A generalized IM calibration strategy is suggested.

N -Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

Comparison of Different Ion Mobility Setups using Poly(ethylene oxide) PEO Polymers: Drift Tube, TIMS and T-Wave

AbstractOver the years, polymer analyses using ion mobility-mass spectrometry (IM-MS) measurements have been performed on different ion mobility spectrometry (IMS) setups. In order to be able to compare literature data taken on different IM(-MS) instruments, ion heating and ion temperature evaluations have already been explored. Nevertheless, extrapolations to other analytes are difficult and thus straightforward same-sample instrument comparisons seem to be the only reliable way to make sure that the different IM(-MS) setups do not greatly change the gas-phase behavior. We used a large range of degrees of polymerization (DP) of poly(ethylene oxide) PEO homopolymers to measure IMS drift times on three different IM-MS setups: a homemade drift tube (DT), a trapped (TIMS), and a traveling wave (T-Wave) IMS setup. The drift time evolutions were followed for increasing polymer DPs (masses) and charge states, and they are found to be comparable and reproducible on the three instruments. Graphical abstractᅟ