Gerhardus J. de Jong

CE-MS in metabolomics

An overview of the use of CE‐MS in the field of metabolomics is provided. Metabolomics is concerned with the comprehensive analysis of endogenous low‐molecular‐weight compounds in biological samples. CE‐MS has demonstrated to be a powerful technique for the profiling of polar metabolites in biological samples. This review covers the use of various CE separation modes, capillary coatings, MS analyzers, sample preparation techniques, and data analysis methods used in CE‐MS for metabolomics. The applicability of CE‐MS in metabolomics research is illustrated by giving examples of the analysis of bacterial extracts, plant extracts, urine, plasma, and cerebrospinal fluid samples. The relevant CE‐MS metabolomics studies published between 2000 and 2008 are presented in tabular form, including information on sample type and pretreatment and MS detection mode. Future developments with regard to the use of alternative ionization techniques, the use of coupled separation systems and the potential of microchip CE systems for metabolomics are discussed.

CE-MS for metabolomics: developments and applications in the period 2014-2016.

This review provides an update of the state‐of‐the‐art of CE‐MS for metabolomic purposes, covering the scientific literature from July 2008 to June 2010. This review describes the different analytical aspects with respect to non‐targeted and targeted metabolomics and the new technological developments used in CE‐MS for metabolomics. The applicability of CE‐MS in metabolomics research is illustrated by examples of the analysis of biomedical and clinical samples, and for bacterial and plant extracts. The relevant papers on CE‐MS for metabolomics are comprehensively summarized in a table, including, e.g. information on sample type and pretreatment, and MS detection mode. Future considerations such as challenges for large‐scale and (quantitative) clinical metabolomics studies and the use of sheathless interfacing and different ionization techniques are discussed.

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.

Wheat Grain Development Is Characterized by Remarkable Trehalose 6-Phosphate Accumulation Pregrain Filling: Tissue Distribution and Relationship to SNF1-Related Protein Kinase1 Activity

Trehalose 6-phosphate (T6P) is a sugar signal that regulates metabolism, growth, and development and inhibits the central regulatory SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11). To better understand the mechanism in wheat (Triticum aestivum) grain, we analyze T6P content and SnRK1 activities. T6P levels changed 178-fold 1 to 45 d after anthesis (DAA), correlating with sucrose content. T6P ranged from 78 nmol g−1 fresh weight (FW) pregrain filling, around 100-fold higher than previously reported in plants, to 0.4 nmol g−1 FW during the desiccation stage. In contrast, maximum SnRK1 activity changed only 3-fold but was inhibited strongly by T6P in vitro. To assess SnRK1 activity in vivo, homologs of SnRK1 marker genes in the wheat transcriptome were identified using Wheat Estimated Transcript Server. SnRK1-induced and -repressed marker genes were expressed differently pregrain filling compared to grain filling consistent with changes in T6P. To investigate this further maternal and filial tissues were compared pre- (7 DAA) and during grain filling (17 DAA). Strikingly, in vitro SnRK1 activity was similar in all tissues in contrast to large changes in tissue distribution of T6P. At 7 DAA T6P was 49 to 119 nmol g−1 FW in filial and maternal tissues sufficient to inhibit SnRK1; at 17 DAA T6P accumulation was almost exclusively endospermal (43 nmol g−1 FW) with 0.6 to 0.8 nmol T6P g−1 FW in embryo and pericarp. The data show a correlation between T6P and sucrose overall that belies a marked effect of tissue type and developmental stage on T6P content, consistent with tissue-specific regulation of SnRK1 by T6P in wheat grain.

Growth Arrest by Trehalose-6-Phosphate: An Astonishing Case of Primary Metabolite Control over Growth by Way of the SnRK1 Signaling Pathway

The strong regulation of plant carbon allocation and growth by trehalose metabolism is important for our understanding of the mechanisms that determine growth and yield, with obvious applications in crop improvement. To gain further insight on the growth arrest by trehalose feeding, we first established that starch-deficient seedlings of the plastidic phosphoglucomutase1 mutant were similarly affected as the wild type on trehalose. Starch accumulation in the source cotyledons, therefore, did not cause starvation and consequent growth arrest in the growing zones. We then screened the FOX collection of Arabidopsis (Arabidopsis thaliana) expressing full-length cDNAs for seedling resistance to 100 mm trehalose. Three independent transgenic lines were identified with dominant segregation of the trehalose resistance trait that overexpress the bZIP11 (for basic region/leucine zipper motif) transcription factor. The resistance of these lines to trehalose could not be explained simply through enhanced trehalase activity or through inhibition of bZIP11 translation. Instead, trehalose-6-phosphate (T6P) accumulation was much increased in bZIP11-overexpressing lines, suggesting that these lines may be insensitive to the effects of T6P. T6P is known to inhibit the central stress-integrating kinase SnRK1 (KIN10) activity. We confirmed that this holds true in extracts from seedlings grown on trehalose, then showed that two independent transgenic lines overexpressing KIN10 were insensitive to trehalose. Moreover, the expression of marker genes known to be jointly controlled by SnRK1 activity and bZIP11 was consistent with low SnRK1 or bZIP11 activity in seedlings on trehalose. These results reveal an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway involving T6P, SnRK1, and bZIP11.

Capillary electrophoresis–mass spectrometry for the analysis of intact proteins 2007–2010

CE coupled to MS has proven to be a powerful analytical tool for the characterization of intact proteins, as it combines the high separation efficiency of CE with the selectivity of MS. This review provides an overview of the development and application of CE‐MS methods within the field of intact protein analysis as published between January 2007 and June 2010. Ongoing technological developments with respect to CE‐MS interfacing, capillary coatings for CE‐MS, coupling of CIEF with MS and chip‐based CE‐MS are treated. Furthermore, CE‐MS of intact proteins involving ESI, MALDI and ICP ionization is outlined and overviews of the use of the various CE‐MS methods are provided by tables. Representative examples illustrate the applicability of CE‐MS for the characterization of proteins, including glycoproteins, biopharmaceuticals, protein–ligand complexes, biomarkers and dietary proteins. It is concluded that CE‐MS is a valuable technique with high potential for intact protein analysis, providing useful information on protein identity and purity, including modifications and degradation products.

CE-MS for metabolomics: Developments and applications in the period 2010-2012: CE and CEC

CE‐MS has emerged as a powerful technique for the profiling of (highly) polar and charged metabolites in biological samples. This review provides an update of the most recent developments in CE‐MS for metabolomics covering the scientific literature from July 2010 to June 2012. The present paper is an update of two previous review papers covering the years 2000–2010 (Electrophoresis 2009, 30, 276–291; Electrophoresis 2011, 32, 52–65). Emerging technological developments used in CE‐MS for metabolomics are discussed, such as the use of novel interfacing techniques for coupling CE to MS. Representative examples illustrate the applicability of CE‐MS in the fields of biomedical, clinical, microbial, plant, environmental and food metabolomics. Concerning targeted and non‐targeted approaches, a comprehensive overview of recent CE‐MS‐based metabolomics studies is given in a table. Information on sample type and pretreatment, capillary coatings and MS detection mode is provided. Finally, general conclusions and perspectives are provided.

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.

CE-MS for the analysis of intact proteins 2010–2012

Since its introduction in 1987, CE‐MS has become an increasingly important technique for the analysis of biomolecules. Since our previous update on CE‐MS methods within the field of intact protein analysis (Electrophoresis 2011, 32, 66–82), a variety of interesting methodological improvements and applications have been reported in literature. Therefore, this article presents an overview of the development and application of CE‐MS for intact protein analysis as published between June 2010 and June 2012. The article is divided in sections that treat CE coupled to MS through ESI, MALDI, and ICP ionization, respectively. In the section about CE‐ESI‐MS, technological developments with respect to CE‐MS interfacing, prevention of protein adsorption, and chip‐based CE‐MS are treated in more detail. Novel interfacing strategies and the development of improved capillary coating strategies appeared to be the major developments. Furthermore, in all sections, the applicability of CE‐MS for intact protein analysis is demonstrated by representative examples, including important developments in the fields of biopharmaceutical characterization and the analysis of proteins in biological samples. Finally, some general conclusions and future perspectives are given.

Ionization techniques in capillary electrophoresis-mass spectrometry: principles, design, and application.

A major step forward in the development and application of capillary electrophoresis (CE) was its coupling to ESI-MS, first reported in 1987. More than two decades later, ESI has remained the principal ionization technique in CE-MS, but a number of other ionization techniques have also been implemented. In this review the state-of-the-art in the employment of soft ionization techniques for CE-MS is presented. First the fundamentals and general challenges of hyphenating conventional CE and microchip electrophoresis with MS are outlined. After elaborating on the characteristics and role of ESI, emphasis is put on alternative ionization techniques including sonic spray ionization (SSI), thermospray ionization (TSI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) and continuous-flow fast atom bombardment (CF-FAB). The principle of each ionization technique is outlined and the experimental set-ups of the CE-MS couplings are described. The strengths and limitations of each ionization technique with respect to CE-MS are discussed and the applicability of the various systems is illustrated by a number of typical examples.