Gold nanoparticle–antibody conjugates for specific extraction and subsequent analysis by liquid chromatography–tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk

Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24 ± 0.04 μg mL⁻¹ GNP suspension for MDA-LDL with an affinity K_d of 5.25 ± 0.11 × 10⁻¹⁰ M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC–MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.     

Syntheses of N10‐substituted 7‐Arylpyrrolo[2,1‐c]‐[1,4]benzodiazepine‐5,11‐diones

Pyrrolo[2,1‐c][1,4]benzodiazepine‐5,11‐dione and its 7‐bromo derivative were alkylated at the N10 atom applying various methods. The resulting products were subjected to Suzuki–Miyaura reactions using a catalyst system consisting of Pd(Cl)₂(PPh₃)₂ and sodium tert‐butanolate in toluene. Results of an X‐ray single crystal analysis are presented.     

Simultaneous separation and analysis of water- and fat-soluble vitamins on multi-modal reversed-phase weak anion exchange material by HPLC-UV

Several methods for the separation of vitamins on HPLC columns were already validated in the last 20 years. However, most of the techniques focus on separating either fat- or water-soluble vitamins and only few methods are intended to separate lipophilic and hydrophilic vitamins simultaneously. A mixed-mode reversed-phase weak anion exchange (RP-WAX) stationary phase was developed in our laboratory in order to address such mixture of analytes with different chemical characteristics, which are difficult to separate on standard columns. The high versatility in usage of the RP-WAX chromatographic material allowed a baseline separation of ten vitamins within a single run, seven water-soluble and three fat-soluble, using three different chromatographic modes: some positively charged vitamins are eluted in ion exclusion and ion repulsion modes whereas the negatively charged molecules are eluted in the ion exchange mechanism. The non-charged molecules are eluted in a classical reversed-phase mode, regarding their polarities. The method was validated for the vitamin analysis in tablets, evaluating selectivity, robustness, linearity, accuracy, and precision. The validated method was finally employed for the analysis of the vitamin content of some commercially available supplement tablets.     

Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review

Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group plays an important role in current analytical chemistry. Application areas range from the preparative enrichment of phospholipids for biotechnological purposes and the separation and purification of plasmid DNA or mRNA to the specific preconcentration of phosphoproteins and -peptides to facilitate their later identification and characterization by mass spectrometry. Most of the recent improvements in this field were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein phosphorylation events with the help of liquid chromatography–mass spectrometry. The high sensitivity of mass spectrometry and the possibility to combine this technique with different separation modes in liquid chromatography have made it the method of choice for proteome analysis. However, in the case of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identification of phosphorylation events. Recent developments in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these limitations. In this review, we will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.     

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Hydrophilic-interaction liquid chromatography (HILIC), reversed-phase chromatography (RPC) and porous graphitic carbon (PGC) chromatography are typically applied for liquid chromatographic separations of protein N-glycans. Hence the performances of these chromatography modes for the separation of fluorescently labeled standard glycan samples (monoclonal antibody, fetuin, ribonuclease-B) covering high-mannose and a broad range of complex type glycans were investigated. In RPC the retention of sialylated glycans was enhanced by adding an ion-pairing agent to the mobile phase, resulting in improved peak shapes for sialylated glycans compared to methods recently reported in literature. For ion pairing RPC (IP-RPC) and HILIC ultra-high performance stationary phases were utilized to maximize the peak capacity and thus the resolution. But due to the shallow gradient in RPC the peak capacity was lower than on PGC. Retention times in HILIC and IP-RPC could be correlated to the monosaccharide compositions of the glycans by multiple linear regression, whereas no adequate model was obtained for PGC chromatography, indicating the significance of the three-dimensional structure of the analytes for retention in this method. Generally low correlations were observed between the chromatography methods, indicating their orthogonality. The high selectivities, as well as the commercial availability of ultra-high performance stationary phases render HILIC the chromatography method of choice for the analysis of glycans. Even though for complete characterization of complex glycan samples a combination of chromatography methods may be necessary.     

Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: a study on mixed-mode retention mechanisms and the pivotal role of solute-silanol interactions in the hydrophilic interaction chromatography elution mode

The separation properties of five silica packings bonded with 1-[3-(trimethoxysilyl)propyl]urea in the range of 0–3.67 μmol m⁻² were investigated in the hydrophilic interaction chromatography (HILIC) elution mode. An increase of the ligand surface density promoted retention of non-charged polar compounds and even more so for acids. An opposite trend was observed for bases, while the amphoteric compound tyrosine exhibited a U-shaped response profile. An overall partitioning retention mechanism was incompatible with these observations; rather, the substantial involvement of adsorptive interactions was implicated. Support for the latter was provided by column-specific changes in analyte retention and concomitant selectivity effects due to variations of salt concentration, type of salt, pH value, organic modifier content, and column temperature. Silica was more selective for separating compounds differing in charge state (e.g. tyramine vs. 4-hydroxybenzoic acid), while in cases where structural differences of solutes resided in non-charged polar groups (e.g. tyramine vs. 5-hydroxydopamine, nucleoside vs. nucleobase) more selective separations were obtained on bonded phases. Hierarchical cluster analysis of the home-made urea-type and three commercial amide-type bonded packings evinced considerable differences in separation properties. The present data emphasise that the role of the packing material under HILIC elution conditions is hardly just the polar support for a dynamic coating with a water-enriched layer. Three major retention mechanisms are claimed to be relevant on bare silica and the urea-type bonded packings: (i) HILIC-type partitioning, (ii) HILIC-type weak adsorption such as hydrogen bonding between solutes and ligands or solutes and silanols (potentially influenced by individual degrees of solvation, salt bridging, etc.), (iii) strong electrostatic (ionic) solute–silanol interactions (attractive/repulsive). Even when non-charged polar bonded phases are used, solute–silanol interactions should not be discounted, which makes them a prime parameter to be characterised by HILIC column tests. Multi/mixed-mode type separations seem to be common under HILIC elution conditions, associated with a great deal of selectivity increments. They are accessible and controllable by a careful choice of the type of packing, the mobile phase composition, and the temperature.     

Static and dynamic binding capacities of human immunoglobulin G on polymethacrylate based mixed-modal, thiophilic and hydrophobic cation exchangers

The aim of this study was to investigate functional increments of ion exchange type ligands, which may improve the performance of mixed-modal ligands for antibody capture out of feed solutions with pH above 6.0 and containing sodium chloride concentrations of 150 mM and higher. For this purpose several functional groups such as sulfonyl, sulfanyl, amide, methoxy, short alkyl and aromatic moieties were tested in combination with a strong sulfonic acid and/or a weak carboxylic acid group. Therefore a series of ligands were synthesized and subsequently coupled onto epoxide activated Fractogel^® EMD. In the first instance, all materials were tested by static binding capacity measurements (SBC) under test conditions, comprising a wide variety of different sodium chloride concentrations and differing pH values ranging from 4.5 to 7.5. From these preliminary experiment it was found that especially the aromatic groups improved the binding of human immunoglobulin G (h-IgG) under isotonic conditions, while other increments, e.g. thiophilic or amide groups, were not able to increase the capacity significantly. Taking the SBC results into account, the most promising materials were investigated under dynamic binding conditions (DBC) with a reduced selection of test conditions (pH 5.5, 6.5 and 7.4 at 75 and 150 mM NaCl). N-benzoyl-homocysteine (material J) and 3,5-dimethoxybenzoyl-homocysteine (material K) showed 100% DBCs of 37 mg/mL and 32 mg/mL in the presence of 75 mM NaCl and pH 6.5. Material L carrying mercaptobenzoic acid as a ligand and tested with the same solution provided a 100% DBC of 68 mg/mL. The influence of Pluronic F68 in a mock feed solution as well as in cell culture supernatant was investigated with the best performing bio-affinity type adsorbent, material L. For the real sample feed subsequent SDS-PAGE was conducted for the collected fractions.