Aspecific Adsorption Research Articles

Surface and Solvent Dependent Adsorption of Three Neuromedin-Like Peptides in Glass and Plastic Syringes

In the last decades, there is an increasing interest in the use of biomolecules, such as peptides, as therapeutic agents. One important challenge inherent to peptides and proteins is the occurrence of aspecific adsorption to all kind of surfaces, hampering drug research and development. In recent years, a lot of research was already performed highlighting adsorption during dissolution, sample pretreatment and analysis. However, only limited data is available about the loss of peptide caused by adsorption to the in vivo administration setup. For this communication we assessed the adsorption of three neuropeptides in glass and plastic syringes by measuring the neuropeptide concentration with nanoUHPLC–MS/MS as a function of time, delivered by the syringe at a flow rate of 2 μL/min. To create a more fundamental approach, three different infusion solvents were examined. Our results clearly show that—although peptide dependent—an important influence of the kind of syringe material and perfusion solvent can be seen on peptide recovery. In addition, we discuss different strategies, and their corresponding in vivo difficulties, to increase peptide recovery during administration.

Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.

Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.

Challenges for the in vivo quantification of brain neuropeptides using microdialysis sampling and LC-MS.

In recent years, neuropeptides and their receptors have received an increased interest in neuropharmacological research. Although these molecules are considered relatively small compared with proteins, their in vivo quantification using microdialysis is more challenging than for small molecules. Low microdialysis recoveries, aspecific adsorption and the presence of various multiply charged precursor ions during ESI-MS/MS detection hampers the in vivo quantification of these low abundant biomolecules. Every step in the workflow, from sampling until analysis, has to be optimized to enable the sensitive analysis of these compounds in microdialysates.

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated.The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments.Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity.Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.

Triple coupled cantilever systems for mass detection and localization

Cantilever sensors have been the subject of a growing attention in the last decades and their use as mass detectors has been proved with attogram sensitivity. Systems of cantilevers on the other hand are still in a preliminary stage and only few devices have been discussed so far. In this paper we propose an original geometry consisting of three mechanically coupled identical cantilevers. We use finite element analysis to investigate the coupling effect on the performance of the system, in particular its mass response. We have fabricated prototypical micron-sized devices, showing that the mass sensitivity of a three coupled cantilever system is comparable to that of a single resonator. We demonstrate that systems of coupled cantilevers offer several advantages over single cantilevers, including less stringent vacuum requirements for operation, mass localization, insensitivity to surface stress and to distributed a-specific adsorption.

Defining protein expression in the urothelium: a problem of more than transitional interest

The transitional epithelium of the bladder, the urothelium, is a challenging tissue to study due to its fragility, complex cellular makeup, stratified composition, and intimate connections to both neural and connective tissue elements. With the increasing focus on the urothelium as a mechanosensory tissue with complex autocrine and paracrine signaling activities, there have arisen a number of unresolved controversies in the urothelial literature regarding whether certain important sensory and signaling proteins are expressed by the urothelium. Prominent examples of this include the transient receptor potential (TRP) family member TRPV1 and the purinergic receptor P2X(3). The problem is more than one of scientific bookkeeping since studies utilizing genetic models (primarily knockout mice) claim additional credibility for urothelial functions when phenotypes are discovered. Furthermore, both of the above-mentioned receptors are important therapeutic targets for various bladder disorders including inflammatory and neuropathic pain. The reasons for the confusion about urothelial expression are manifold, but they likely include low expression levels in some cases, poor specificity of antibodies (sometimes lacking adequate controls), the presence of nonurothelial cells resident within the urothelium, and the fact that the urothelium is particularly prone to aspecific adsorption of antibodies. In this review, we attempt to summarize some of the pitfalls with currently accepted practices in this regard, as well as to describe a set of guidelines which will improve the reliability of conclusions related to urothelial expression. It is hoped that this will be of value to investigators studying the urothelium, to those attempting to interpret conflicts in the literature, and hopefully also those charged with reviewing unpublished work. These recommendations will outline a set of "baseline" and "best practice" guidelines by which both researchers and reviewers will be able to evaluate the evidence presented.

Pitfalls and solutions in assaying anandamide transport in cells

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.

Fibre-entrapped antibodies

Anti-insulin and anti-HGH serum were entrapped in cellulose triacetate fibres without alteration of their biological properties by spinning solvents. The aspecific adsorption of insulin by cellulose triacetate fibres is low and time independent. Anti-insulin serum entrapped in fibres binds insulin with high yields and its capture depends on the contact time. The equilibrium constant of the reaction antigen-antibody and the number of antibody sites are not modified substantially by the immobilization procedure. The HGH uptake by anti-HGH serum entrapped in fibres is a linear function of HGH concentration in the solution.

Amino acid incorporation by human milk fat globules membranes (author's transl)

Human milk fat globule membranes (MFGM) can incorporate radioactive 14C amino acids in a hot trichloracetic acid-insoluble material. Aspecific adsorption and bacterial contamination are unlikely. The products of protein synthesis were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by action of proteolytic enzymes. Various inhibitors of protein synthesis were assayed. Fragments of rough endoplasmic reticulum or mitochondria could be involved in this incorporation.

SECOND ANTIBODY CHEMICALLY LINKED TO CELLULOSE FOR THE SEPARATION OF BOUND AND FREE HORMONE: AN IMPROVEMENT OVER SOLUBLE SECOND ANTIBODY IN GONADOTROPHIN RADIOIMMUNOASSAY

Coupling the second antibody to a solid pase (DASP) was found to be a definite improvement over the classical technique of soluble second antibody (DA) separation of bound and free hormone in the radioimmunoassays of gonadotrophins in plasma or in serum. The duration (2.5 to 7.5 min) and the pH (10.5 to 12.5) of the cyanogen bromide activation of microcristalline cellulose did not affect the coupling capacity or the immunoreactivity of the second antibody, nor did it augment aspecific adsorption of free gonadotrophin on the cellulose matrix. Precision of duplicates was comparable for both methods but the accuracy and the detection limit of the solid phase system were higher due to a better separation of bound and free hormone, to the lower blank value and to the absence of aspecific interference of protein concentration. Cost was lower since much less second antibody was needed in the solid phase system than in the soluble system and since the experimental procedures were simplified by the absence of prozoning effect and the solid nature of the cellulose matrix.