CE-MS for metabolomics: Developments and applications in the period 2016-2018.

In the field of metabolomics, CE-MS is now recognized as a strong analytical technique for the analysis of (highly) polar and charged metabolites in a wide range of biological samples. Over the past few years, significant attention has been paid to the design and improvement of CE-MS approaches for (large-scale) metabolic profiling studies and for establishing protocols in order to further expand the role of CE-MS in metabolomics. In this paper, which is a follow-up of a previous review paper covering the years 2014-2016 (Electrophoresis 2017, 38, 190-202), main advances in CE-MS approaches for metabolomics studies are outlined covering the literature from July 2016 to June 2018. Aspects like developments in interfacing designs and data analysis tools for improving the performance of CE-MS for metabolomics are discussed. Representative examples highlight the utility of CE-MS in the fields of biomedical, clinical, microbial, and plant metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings and MS detection mode. Finally, some general conclusions and perspectives are given.

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors.

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd ) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea.

Chiral capillary electrophoresis with UV-excited fluorescence detection for the enantioselective analysis of 9-fluorenylmethoxycarbonyl-derivatized amino acids.

The potential of capillary electrophoresis (CE) with ultraviolet (UV)-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was investigated. DL-AAs were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC)-Cl to allow their fluorescence detection and enhance enantioseparation. Fluorescence detection was achieved employing optical fibers, leading UV excitation light (< 300 nm) from a Xe-Hg lamp to the capillary window, and fluorescence emission to a spectrograph equipped with a charge-coupled device (CCD). Signal averaging over time and emission wavelength intervals was carried out to improve the signal-to-noise ratio of the FMOC-AAs. A background electrolyte (BGE) of 40 mM sodium tetraborate (pH 9.5), containing 15% isopropanol (v/v), 30 mM sodium dodecyl sulfate (SDS), and 30 mM ß-cyclodextrin (ß-CD), was found optimal for AA chemo- and enantioseparation. Enantioresolutions of 1.0 or higher were achieved for 16 proteinogenic DL-AAs. Limits of detection (LODs) were in the 10-100-nM range (injected concentration) for the D-AA enantiomers, except for FMOC-D-tryptophan (536 nM) which showed intramolecular fluorescence quenching. Linearity (R2 > 0.997) and repeatability for peak height (relative standard deviations (RSDs) < 7.0%; n = 5) and electrophoretic mobility (RSDs < 0.6%; n = 5) of individual AA enantiomers were established for chiral analysis of DL-AA mixtures. The applicability of the method was investigated by the analysis of cerebrospinal fluid (CSF). Next to L-AAs, endogenous levels of D-glutamine and D-aspartic acid could be measured in CSF revealing enantiomeric ratios of 0.35 and 19.6%, respectively. This indicates the method's potential for the analysis of low concentrations of D-AAs in presence of abundant L-AAs.

Enantioselective micellar electrokinetic chromatography of dl-amino acids using (+)-1-(9-fluorenyl)-ethyl chloroformate derivatization and UV-induced fluorescence detection.

Chiral analysis of dl-amino acids was achieved by micellar electrokinetic chromatography coupled with UV-excited fluorescence detection. The fluorescent reagent (+)-1-(9-fluorenyl)ethyl chloroformate was employed as chiral amino acid derivatizing agent and sodium dodecyl sulfate served as pseudo-stationary phase for separating the formed amino acid diastereomers. Sensitive analysis of (+)-1-(9-fluorenyl)ethyl chloroformate-amino acids was achieved applying a xenon-mercury lamp for ultraviolet excitation, and a spectrograph and charge-coupled device for wavelength-resolved emission detection. Applying signal integration over a 30 nm emission wavelength interval, signal-to-noise ratios for derivatized amino acids were up to 23 times higher as obtained using a standard photomultiplier for detection. The background electrolyte composition (electrolyte, pH, sodium dodecyl sulfate concentration, and organic solvent) was studied in order to attain optimal chemo- and enantioseparation. Enantioseparation of 12 proteinogenic dl-amino acids was achieved with chiral resolutions between 1.2 and 7.9, and detection limits for most derivatized amino acids in the 13-60 nM range (injected concentration). Linearity (coefficients of determination > 0.985) and peak-area and migration-time repeatabilities (relative standard deviations lower than 2.6 and 1.9%, respectively) were satisfactory. The employed fluorescence detection system provided up to 100-times better signal-to-noise ratios for (+)-1-(9-fluorenyl)ethyl chloroformate-amino acids than ultraviolet absorbance detection, showing good potential for d-amino acid analysis.

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

CE-MS can be considered a useful analytical technique for the global profiling of (highly) polar and charged metabolites in various samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolomics studies. In this paper, which is a follow-up of a previous review paper covering the years 2012-2014 (Electrophoresis 2015, 36, 212-224), recent CE-MS strategies developed for metabolomics covering the literature from July 2014 to June 2016 are outlined. Attention will be paid to new CE-MS approaches for the profiling of anionic metabolites and the potential of SPE coupled to CE-MS is also demonstrated. Representative examples illustrate the applicability of CE-MS in the fields of biomedical, clinical, microbial, plant, and food metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given.

Developments in coupled solid-phase extraction-capillary electrophoresis 2013-2015.

An overview of the design and application of coupled solid-phase extraction-capillary electrophoresis (SPE-CE) systems reported in the literature between July 2013 and June 2015 is provided in this paper. The present article is a continuation of our previous review papers on this topic which covered the time period 2000-2013 (Electrophoresis 2008, 29, 108-128; Electrophoresis 2010, 31, 44-54; Electrophoresis 2012, 33, 243-250; Electrophoresis 2014, 35, 128-137). The use of in-line and on-line SPE-CE approaches is treated and outlined in this review. Recent advancements, such as, for example, the use of aptamers as affinity material for in-line SPE-CE, the use of a bead string design for in-line fritless SPE-CE, and new interfacing techniques for the on-line coupling of SPE to CE, are outlined. Selected examples demonstrate the applicability of the coupled SPE-CE systems for biomedical, pharmaceutical, environmental, and food studies. A complete overview of the recent SPE-CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and perspectives are provided.

Enantioselective analysis of proteinogenic amino acids in cerebrospinal fluid by capillary electrophoresis-mass spectrometry.

d-Amino acids (AAs) are increasingly being recognized as essential molecules in biological systems. Enantioselective analysis of proteinogenic AAs in biological samples was accomplished by CE-MS employing ß-CD as chiral selector and ESI via sheath-liquid (SL) interfacing. Prior to analysis, AAs were fully derivatized with FMOC, improving AA-enantiomer separation and ESI efficiency. In order to optimize the separation and MS detection of FMOC-AAs, the effects of type and concentration of CD in the BGE, the composition of the SL, and MS-interfacing parameters were evaluated. Using a BGE of 10 mM ß-CD in 50 mM ammonium bicarbonate (pH 8) containing 15% v/v isopropanol, a SL of isopropanol-water-1 M ammonium bicarbonate (50:50:1, v/v/v) at a flow rate of 3 µL/min, and a nebulizer gas pressure of 2 psi, 15 proteinogenic AAs could be detected with enantioresolutions up to 3.5 and detection limits down to 0.9 µM (equivalent to less than 3 pg AA injected). The selectivity of the method was demonstrated by the analysis of spiked cerebrospinal fluid, allowing specific detection of d-AAs. Repeatability and linearity obtained for cerebrospinal fluid were similar to standard solutions, with peak area and migration-time RSDs (n = 5) below 16.2 and 1.6%, respectively, and a linear response (R(2) ≥ 0.977) in the 3-90 µM range.

CE-MS for metabolomics: developments and applications in the period 2012-2014.

In the field of metabolomics, CE-MS is now regarded as a useful complementary analytical technique for the profiling of (highly) polar ionogenic metabolites in biological samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolic profiling studies. This paper, which is a follow-up of three previous review papers covering the years 2000-2012 [Electrophoresis 2009, 30, 276-291; Electrophoresis 2011, 32, 52-65; Electrophoresis 2013, 34, 86-98], provides an update of these developments covering the scientific literature from July 2012 to June 2014. Attention will be paid to novel interfacing techniques for coupling CE to MS and their implications for metabolomics studies. The potential of CEC-MS and MEKC-MS are also considered, and CE-MS systems for high-throughput metabolic profiling are discussed. The applicability of CE-MS for metabolomics studies is demonstrated by representative examples in the fields of biomedical, clinical, microbial, plant, environmental, and food metabolomics. An overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given.

Developments in coupled solid-phase extraction-capillary electrophoresis 2011-2013.

This article presents an overview of the design and application of coupled SPE-CE systems that have been reported in the literature between January 2011 and June 2013. The present paper is an update of three previous review papers covering the years 2000-2011 (Electrophoresis 2008, 29, 108-128; Electrophoresis 2010, 31, 44-54; Electrophoresis 2012, 33, 243-250). The use of in-line and on-line SPE-CE approaches is described in this review. Emerging technological developments, such as the use of in-line frit-free SPE and chip-based SPE for extraction of sample components prior to CE analysis, are outlined. Selected examples illustrate the applicability of SPE-CE in biomedical, pharmaceutical, and environmental analysis. A complete overview of recent SPE-CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and future perspectives are provided.

Evaluation of fritless solid-phase extraction coupled on-line with capillary electrophoresis-mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid.

Fritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 µm id was packed with a C18 sorbent (particle size > 50 µm), which was retained between a short inlet capillary and a separation capillary (50 µm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.

Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins.

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-ß allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

CE-MS for metabolomics: developments and applications in the period 2010-2012.

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.

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.

Micellar electrokinetic chromatography-electrospray ionization mass spectrometry employing a volatile surfactant for the analysis of amino acids in human urine.

A new MEKC-ESI-MS method for the analysis of amino acids (AAs) in human urine was developed employing ammonium perfluorooctanoate (APFO) as volatile surfactant. The influence of APFO on the MS signal of AAs was evaluated by infusion experiments, which showed that APFO hardly affects analyte responses and presents significantly less ion suppression than equal concentrations of ammonium acetate. In order to obtain efficient separation of AAs, MEKC parameters such as the pH and APFO concentration of the BGE, were optimized. Optimum AA resolution, including baseline separation of leucine and isoleucine, was obtained using 150 mM APFO (pH 9.0) as BGE, representing a considerable selectivity improvement over CE using 50 mM ammonium acetate (pH 9.0). Optimization of CE-MS parameters, such as sheath liquid composition and flow rate, and ESI and MS settings, led to LODs ranging from 9 to 26 ng/mL for the 20 tested AAs, which is highly favorable for an MEKC-MS method. Good linearity (r(2) > 0.99) and repeatability were obtained for all AAs tested with RSD values of 3.0-6.7% for peak area and <1.5% for migration time. The applicability of the MEKC-MS method was demonstrated by the quantitative determination of AAs in urine employing only a 1:1 dilution with BGE as sample pretreatment. All AAs could selectively be detected and quantified obtaining relevant concentration values for normal human urine.

Potential of capillary electrophoresis with wavelength-resolved fluorescence detection for protein unfolding studies using ß-lactoglobulin B as a test compound.

Capillary electrophoresis (CE) with wavelength-resolved fluorescence detection (wrFlu) was evaluated for the study of protein unfolding using non-reduced and reduced ß-lactoglobulin B (ß-LGB) as model compounds. Protein unfolding was achieved by incubation in sodium phosphate (pH 3.0) containing increasing concentrations of urea (0-7.1 M). CE-wrFlu was performed using the incubation media as background electrolytes (BGEs). At low urea concentrations (0-3.1 M), CE-wrFlu analysis of non-reduced ß-LGB showed a single peak with a maximum emission wavelength (λmax) of 333 nm, indicating the protein was in its folded state. When ß-LGB was exposed to 3.6 and 4.1 M urea, CE-wrFlu revealed an additional peak with a λmax of 353 nm and a reduced migration time, suggesting the formation of fully unfolded species. Upon further raising the urea concentration up to 6.5 M, the peak intensity of the unfolded protein increased. At the same time, the later-migrating peak became wider and lower, showing a decrease of migration time and a shift of λmax (333-353 nm), indicating gradual unfolding. Construction of a λmax-based transition curve for the later-migrating ß-LGB species provided a denaturant-concentration midpoint of unfolding (cm) of 5.39 M, which was similar to the cm determined by plotting the corrected effective electrophoretic mobility (µeff,c) vs. urea concentration. Stand-alone fluorescence spectroscopy of the same ß-LGB solutions revealed a lower cm (4.97 M), most probably because the respective ß-LGB species were not separated, yielding ensemble average data. For reduced ß-LGB, at all tested urea concentrations one protein peak was observed, whereas λmax and µeff,c indicated protein unfolding at much lower urea concentrations (cm of 1.2 M). We conclude that CE-wrFlu can distinguish protein conformational states and thus may provide useful additional information on unfolding pathways.

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis-mass spectrometry.

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9-13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

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.

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.

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.

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection.

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal-to-noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE-Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO-α and EPO-ß) based on the recorded glycoform pattern. The developed CE-Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.