Gold nanoparticle-conjugated anti-oxidized low-density lipoprotein antibodies for targeted lipidomics of oxidative stress biomarkers.

Oxidized low-density lipoproteins (OxLDLs), in particular, oxidized phosphatidylcholines (OxPCs), are known to be involved in pathophysiological processes such as cardiovascular diseases and are described as potential biomarkers, for example, for atherosclerosis. In our study, we used the specific affinity of anti-OxLDL antibodies (Abs) conjugated to gold nanoparticles (GNPs) for extraction and enrichment of OxPCs via selective trapping of OxLDLs from plasma combined with the sensitive detection by liquid chromatography/tandem-mass spectrometry (LC-MS/MS). Successful bioconjugation chemistry of Abs via a bifunctional polyethylene glycol (PEG) spacer and protein G linkage, respectively, was controlled by measuring the surface plasmon resonance (SPR) spectra, size, and zeta potentials. Furthermore, the amount of Ab immobilized onto GNP via the PEG linker was determined. With the optimized immobilization chemistry, the ability and potential of the GNP-based extraction procedure was used for the determination of the dissociation constant, K(d), of the OxLDL binding to the GNP-Ab conjugate. Moreover, apparent K(d)'s were determined for individual PCs and their oxidation products using the compound-specific selected reaction monitoring mode, which allows the characterization of the Ab affinity and, thus, assessment of the potential antigenicity of (Ox)PCs bound to OxLDLs. In summary, the application of GNP-based bioanalysis for selective targeting of OxLDLs and the fast and sensitive detection by LC-MS/MS offers new possibilities for targeted lipidomics in lipoproteins as well as for oxidative stress lipid biomarker screening.

Comparative method evaluation for size and size-distribution analysis of gold nanoparticles.

Gold nanoparticles (GNPs) are popular colloidal substrates in various sensor, imaging, and nanomedicine applications. In separation science, they have raised some interest as a support for sample preparation. Reasons for their popularity are their low cost, ability for size-controlled synthesis with well-defined narrow nanoparticle size distributions, as well as straightforward surface functionalization by self-assembling (thiol-containing) molecules on the surface, which allows flexible introduction of functionalities for the selective capture of analytes. Most commonly, the method of first choice for size determination is dynamic light scattering (DLS). However, DLS has some serious shortcomings, and results from DLS may be misleading. For this reason, in this contribution several distinct complementary nanoparticle sizing methodologies were utilized and compared to characterize citrate-capped GNPs of different diameters in the range of 13-26 nm. Weaknesses and strengths of DLS, transmission electron microscopy, asymmetrical-flow field-flow fractionation and nanoelectrospray gas-phase electrophoretic mobility molecular analysis are discussed and the results comparatively assessed. Furthermore, the distinct GNPs were characterized by measuring their zeta-potential and surface plasmon resonance spectra. Overall, the combination of methods for GNP characterization gives a more realistic and comprehensive picture of their real physicochemical properties, (hydrodynamic) diameter, and size distribution.

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography.

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and ß-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.

Plasma Proteomics for Epidemiology: Increasing Throughput With Standard-Flow Rates.

BACKGROUND: Mass spectrometry is selective and sensitive, permitting routine quantification of multiple plasma proteins. However, commonly used nanoflow liquid chromatography (LC) approaches hamper sample throughput, reproducibility, and robustness. For this reason, most publications using plasma proteomics to date are small in study size. METHODS AND RESULTS: Here, we tested a standard-flow LC mass spectrometry (MS) method using multiple reaction monitoring for the application to large epidemiological cohorts. We have reduced the LC-MS run time to almost a third of the nanoflow LC-MS approach. On the basis of a comparison of the quantification of 100 plasma proteins in >1500 LC-MS runs, the SD range of the retention time during continuous operation was substantially lower with the standard-flow LC-MS (<0.05 minutes) compared with the nanoflow LC-MS method (0.26-0.44 minutes). In addition, the standard-flow LC method also offered less variation in protein measurements. However, 5× more sample volume was required to achieve similar sensitivity. Two different commercial multiple reaction monitoring kits and an antibody-based multiplexing kit were used to compare the apolipoprotein measurements in a subset of samples. In general, good agreement was observed between the 2 multiple reaction monitoring kits, but some of the multiple reaction monitoring-based measurements differed from antibody-based assays. CONCLUSIONS: The multiplexing capability of LC-MS combined with a standard-flow method increases throughput and reduces the costs of large-scale protein measurements in epidemiological cohorts, but protein rather than peptide standards will be required for defined absolute proteoform quantification.

Lipidomics: quest for molecular lipid biomarkers in cardiovascular disease.

Lipidomics is the comprehensive analysis of molecular lipid species, including their quantitation and metabolic pathways. The huge diversity of native lipids and their modifications make lipidomic analyses challenging. The method of choice for sensitive detection and quantitation of molecular lipid species is mass spectrometry, either by direct infusion (shotgun lipidomics) or coupled with liquid chromatography. Although shotgun lipidomics allows for high-throughput analysis, low-abundant lipid species are not detected. Previous separation of lipid species by liquid chromatography increases ionization efficiency and is better suited for quantifying low abundant and isomeric lipid species. In this review, we will discuss the potential of lipidomics for cardiovascular research. To date, cardiovascular research predominantly focuses on the role of lipid classes rather than molecular entities. An in-depth knowledge about the molecular lipid species that contribute to the pathophysiology of cardiovascular diseases may provide better biomarkers and novel therapeutic targets for cardiovascular disease.

Quantifying thiol ligand density of self-assembled monolayers on gold nanoparticles by inductively coupled plasma-mass spectrometry.

Gold nanoparticles (GNPs) are often used as colloidal carriers in numerous applications owing to their low-cost and size-controlled preparation as well as their straightforward surface functionalization with thiol containing molecules forming self-assembling monolayers (SAM). The quantification of the ligand density of such modified GNPs is technically challenging, yet of utmost importance for quality control in many applications. In this contribution, a new method for the determination of the surface coverage of GNPs with thiol containing ligands is proposed. It makes use of the measurement of the gold-to-sulfur (Au/S) ratio by inductively coupled plasma mass spectrometry (ICP-MS) and its dependence on the nanoparticle diameter. The simultaneous ICP-MS measurement of gold and sulfur was carefully validated and found to be a robust method with a relative standard uncertainty of lower than 10%. A major advantage of this method is the independence from sample preparation; for example, sample loss during the washing steps is not affecting the results. To demonstrate the utility of the straightforward method, GNPs of different diameters were synthesized and derivatized on the surface with bifunctional (lipophilic) ω-mercapto-alkanoic acids and (hydrophilic) mercapto-poly(ethylene glycol) (PEG)(n)-carboxylic acids, respectively, by self-assembling monolayer (SAM) formation. Thereby, a size-independent but ligand-chain length-dependent ligand density was found. The surface coverage increases from 4.3 to 6.3 molecules nm⁻² with a decrease of ligand chain length from 3.52 to 0.68 nm. Furthermore, no significant difference between the surface coverage of hydrophilic and lipophilic ligands with approximately the same ligand length was found, indicating that sterical hindrance is of more importance than, for example, intermolecular strand interactions of Van der Waals forces as claimed in other studies.

Bioconjugation of trypsin onto gold nanoparticles: effect of surface chemistry on bioactivity.

The systematic study of activity, long-time stability and auto-digestion of trypsin immobilized onto gold nanoparticles (GNPs) is described in this paper and compared to trypsin in-solution. Thereby, the influence of GNP's size and immobilization chemistry by various linkers differing in lipophilicity/hydrophilicity and spacer lengths was investigated with regard to the bioactivity of the conjugated enzyme. GNPs with different sizes were prepared by reduction and simultaneous stabilization with trisodium citrate and characterized by UV/vis spectra, dynamic light scattering (DLS), ζ-potential measurements and transmission electron microscopy (TEM). GNPs were derivatized by self-assembling of bifunctional thiol reagents on the nanoparticle (NP) surface via dative thiol-gold bond yielding a carboxylic acid functionalized surface. Trypsin was either attached directly via hydrophobic and ionic interactions onto the citrate stabilized GNPs or immobilized via EDC/NHS bioconjugation onto the carboxylic functionalized GNPs, respectively. The amount of bound trypsin was quantified by measuring the absorbance at 280 nm. The activity of bound enzyme and its Michaelis Menten kinetic parameter K(m) and v(max) were measured by the standard chromogenic substrate N(α)-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA). Finally, digestion of a standard protein mixture with the trypsin-conjugated NPs followed by analysis with LC-ESI-MS and successful MASCOT search demonstrated the applicability of the new heterogenous nano-structured biocatalyst. It could be shown that the amount of immobilized trypsin and its activity can be increased by a factor of 6 using a long hydrophilic spacer with simultaneous reduced auto-digestion and reduced digestion time. The applicability of the new trypsin bioreactor was proven by digestion of casein and identification of α- as well as κ-casein by subsequent MASCOT search.