Structure and Dynamics of Human Chemokine CCL16-Implications for Biological Activity.

Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.

Novel RP-HPLC based assay for selective and sensitive endotoxin quantification.

The paper presents a novel instrumental analytical endotoxin quantification assay. It uses common analytical laboratory equipment (HPLC-FLD) and allows quantifying endotoxins (ETs) in different matrices from about 109 EU per mL down to about 40 EU per mL (RSE based). Test results are obtained in concentration units (e.g. ng ET per mL), which can then be converted to commonly used endotoxin units (EU per mL) in case of known pyrogenic activity. During endotoxin hydrolysis, the endotoxin specific rare sugar acid KDO is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-HPLC followed by KDO-DMB quantification with a fluorescence detector. Based on the KDO content, the endotoxin content in the sample is calculated. The developed assay is economic and has a small error. Its applicability was demonstrated in applied research. ETs were quantified in purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results obtained for the same samples. A high correlation was found between the results of both methods. Further, the new assay was utilized with high success during the development of novel endotoxin specific depth filters, which allow efficient, economic and sustainable ET removal during DSP. Those examples demonstrate that the new assay has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the major health authorities worldwide for the release of commercial pharmaceutical products.

Capillary electrophoresis-mass spectrometry for the direct analysis of glyphosate: method development and application to beer beverages and environmental studies.

In this study, we developed and validated a CE-TOF-MS method for the quantification of glyphosate (N-(phosphonomethyl)glycine) and its major degradation product aminomethylphosphonic acid (AMPA) in different samples including beer, media from toxicological analysis with Daphnia magna, and sorption experiments. Using a background electrolyte (BGE) of very low pH, where glyphosate is still negatively charged but many matrix components become neutral or protonated, a very high separation selectivity was reached. The presence of inorganic salts in the sample was advantageous with regard to preconcentration via transient isotachophoresis. The advantages of our new method are the following: no derivatization is needed, high separation selectivity and thus matrix tolerance, speed of analysis, limits of detection suitable for many applications in food and environmental science, negligible disturbance by metal chelation. LODs for glyphosate were < 5 µg/L for both aqueous and beer samples, the linear range in aqueous samples was 5-3000 µg/L, for beer samples 10-3000 µg/L. For AMPA, LODs were 3.3 and 30.6 µg/L, and the linear range 10-3000 µg/L and 50-3000 µg/L, for aqueous and beer samples, respectively. Recoveries in beer samples for glyphosate were 94.3-110.7% and for AMPA 80.2-100.4%. We analyzed 12 German and 2 Danish beer samples. Quantification of glyphosate and AMPA was possible using isotopically labeled standards without enrichment, purification, or dilution, only degassing and filtration were required for sample preparation. Finally, we demonstrate the applicability of the method for other strong acids, relevant in food and environmental sciences such as N-acetyl glyphosate, N-acetyl AMPA (present in some glyphosate resistant crop), trifluoroacetic acid, 2-methyl-4-chlorophenoxyacetic acid, glufosinate and its degradation product 3-(methylphosphinico)propionic acid, oxamic acid, and others.

Novel approach for the synthesis of a neutral and covalently bound capillary coating for capillary electrophoresis-mass spectrometry made from highly polar and pH-persistent N-acryloylamido ethoxyethanol.

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).

Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE-MS Peptide Analysis: Strategies and Implementation.

Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aß peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings.

Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish.

Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.

Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS.

In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-ß-peptide (Aß) associated with Alzheimer's disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.

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.

Column coupling isotachophoresis-capillary electrophoresis with mass spectrometric detection: characterization and optimization of microfluidic interfaces.

Two-dimensional electrophoretic separations are one of the most promising tools for the continuously growing needs of different bioanalytical fields such as proteomics and metabolomics. In this work we present the design and the implementation of a two-dimensional electrophoretic separation coupled to mass spectrometry. We started our work studying the sample transfer characteristics of different microfluidic interfaces compatible with capillary coupling for two-dimensional electrophoretic separations. These junctions are aimed at method decoupling and sample transfer in a modular two-dimensional electrophoretic separation system. In order to perform the characterization of the interfaces, we carried out capillary electrophoresis experiments and numerical simulations using three cationic compounds under different flow conditions. The comparison of the experimental and simulation results enables us to clearly define the desirable characteristics of interfaces in order to achieve method orthogonality with lossless sample transfer in a two-dimensional separation system. Finally, we present a glass microfluidic chip as interface for the implementation of a novel hybrid modular system for performing two-dimensional electrophoretic separations involving isotachophoresis and capillary electrophoresis. In this setup we include mass spectrometric and contactless capacitively coupled conductivity detection to monitor the separation process. We demonstrate the ability of the setup to be used as a flexible analysis tool by performing preconcentration, separation, detection and identification of four different human angiotensin peptides.

Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings.

Capillary coatings are crucial for high-quality separation performance in capillary electrophoresis analysis of proteins or peptides as they prevent analyte adsorption at the capillary wall. These coating materials have to fulfill many requirements such as a good separation performance and ensuring a good repeatability. The number of commercially available coating materials is still limited, especially with regard to the charge density on the coating material and the induced electroosmotic flow (EOF) velocity. In this work, we compare the separation performance of the novel self-made cationic capillary coating OHNOON and two commercially available coating materials, the acrylamide based, neutral LN® and the cationic hexadimethrine bromide (Polybrene), using the same coating procedure for all three coating materials. The coatings are investigated regarding the separation efficiency, analyte resolution, coating stability, and migration time stability in tryptic peptide analysis. Good separation performance was confirmed for all three coating materials: all coatings provided high plate numbers of up to 400,000-500,000 and a repeatability of the EOF and the analyte migration times in the range of 1% relative standard deviation or below. Our results reveal a moderate EOF velocity for the novel OHNOON coating in comparison to the Polybrene coating. We present a detailed discussion of the impact of this reduced EOF velocity and the separation performance. The results presented here will help to define the necessary properties of coating materials to achieve the best compromise between speed of analysis and resolution for the respective application. We show that our novel OHNOON coating is especially valuable for the analysis of low mobility analytes and for samples with a broad range of analyte mobilities.

Determination of the interparticle void volume in packed beds via intraparticle Donnan exclusion.

Interparticle void volumes and porosities of packed capillaries have been determined using intraparticle Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The operational domain of this approach has been characterized for bare silica, reversed-phase, and strong cation-exchange materials (with different particle sizes and intraparticle pore sizes) in dependence of the mobile phase ionic strength. Interparticle porosities agree well with those analyzed by inverse size-exclusion chromatography (ISEC). Limitations to the use of Donnan exclusion (electrostatic exclusion) and ISEC (mechanical exclusion) arise as either type of exclusion becomes noticeable also in the cusp regions between the particles, or as the intraparticle pores are so large that complete electrostatic and size-exclusion are difficult to realize. Our data confirm that intraparticle Donnan exclusion presents a most simple, fast, and reliable approach for the analysis of packing densities.