Martin Kratzmeier

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1

Abstract Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.

Molecular weight determination of high molecular mass (glyco)proteins using CGE-on-a-chip, planar SDS-PAGE and MALDI-TOF-MS†

The molecular weights (MW) of seven (glyco)proteins, of which five were plasma‐derived, with MWs higher than 200 kDa were determined with three techniques: CGE‐on‐a‐chip, SDS‐PAGE and MALDI‐TOF‐MS. While the analysis of medium to high MW proteins with SDS‐PAGE was an already well‐established technique, the usefulness of MALDI‐TOF‐MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE‐on‐a‐chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE‐on‐a‐chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS‐PAGE and 660 kDa in the case of the high MW CGE‐on‐a‐chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI‐TOF‐MS provided accurate MWs for all high MW proteins up to 1 MDa.

Characterization of cross-linked gelatin nanoparticles by electrophoretic techniques in the liquid and the gas phase

Biodegradable nanoparticles (NPs) and hence, for example, NPs prepared from glutaraldehyde cross‐linked gelatin (gelatin NPs) are lately receiving increased attention in various fields such as pharmaceutical technology and nutraceutics as biocompatible carriers for hardly water soluble drugs, molecules intended for sustained release or targeted transport. However, in vivo application of such materials requires a thoroughly characterization of corresponding particles. In a previous manuscript, we demonstrated the applicability of chip electrophoresis for the separation of gelatin NPs from NP building blocks. Following our previous results, we intensified our efforts in the characterization of gelatin NPs by electrophoresis in the liquid (capillary and chip format) and the gas phase (gas phase electrophoretic mobility molecular analysis). In doing so, we demonstrated differences between nominally comparable (from the concentration of initially employed material for cross‐linking) gelatin NP preparation batches concerning (i) the amount of obtained NPs, (ii) the degree of NP cross‐linking, (iii) the amount of NP building blocks present within samples, and (iv) the electrophoretic mobility diameter of NPs. Differences were even more pronounced when NP preparations from batches with different content of initially employed gelatin were compared. Additionally, we compared three setups for the removal of low molecular weight components from samples after fluorescence labeling of NPs. In overall, the combination of the three employed analytical methods for gelatin NP characterization—CE in the capillary and the chip format as well as gas phase electrophoretic mobility molecular analysis—allows a more thoroughly characterization of NP containing samples.

Challenges of glycoprotein analysis by microchip capillary gel electrophoresis

Glycosylations severely influence a proteins biological and physicochemical properties. Five exemplary proteins with varying glycan moieties were chosen to establish molecular weight (MW) determination (sizing), quantitation, and sensitivity of detection for microchip capillary gel electrophoresis (MCGE). Although sizing showed increasing deviations from literature values (SDS‐PAGE or MALDI‐MS) with a concomitant higher degree of analyte glycosylation, the reproducibility of MW determination and accuracy of quantitation with high sensitivity and reliability were demonstrated. Additionally, speed of analysis together with the low level of analyte consumption render MCGE attractive as an alternative to conventional SDS‐PAGE.

Simple bead assay for detection of live bacteria (Escherichia coli)

Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.

Detection of specific strains of viable bacterial pathogens by using RNA bead assays and flow cytometry with 2100 Bioanalyzer.

Bead assays are an emerging microbial detection technology with the capability for rapid detection of extremely low levels of viable pathogens. Such technologies are of high value in clinical settings and in the food industry. Here, we perform a bead assay for extracted 16S rRNA from Escherichia coli (strain K12) with the flow cytometry readout on a 2100 Bioanalyzer, a highly accurate, small-scale flow cytometer system.

Microchip capillary gel electrophoresis combined with lectin affinity enrichment employing magnetic beads for glycoprotein analysis

AbstractDue to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstractGlycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides