Mechanistic investigation of the on-surface enzymatic digestion (oSED) protein adsorption detection method using targeted mass spectrometry.

This study describes our efforts to study some of the mechanistic aspects of the earlier established on-surface enzymatic digestion (oSED) method. In a multitude of application areas, it has become important to be able to fully characterize and understand selective protein adsorption to biomaterial surfaces for various applications, including biomedicine (implants), nanotechnology (microchip surfaces and sensors) and materials sciences. Herein, the investigation of the mechanistic aspects was based on microdialysis catheter tubes that were flushed with controlled protein solutions mimicking the extracellular fluid of the brain. The protein adsorption properties were monitored using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) with a targeted method. The temporally resolved results show that most proteins stay adsorbed onto the surface during the entire digestion process and are only cut away piece by piece, whereas smaller proteins and peptides seem to desorb rather easily from the surface. This information will simplify the interpretation of data generated using the oSED method and can also be used for the characterization of the physicochemical properties controlling the adsorption of individual proteins to specific surfaces.

Influence of surface modification and static pressure on microdialysis protein extraction efficiency.

There is growing interest in using microdialysis (MD) for monitoring larger and more complex molecules such as neuropeptides and proteins. This promotes the use of MD membranes with molecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of the membrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure. In the meantime, despite the large pore size, studies have shown that membrane biofouling still lead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextran and Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency (EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests, using as MD sample a protein standard with similar concentrations as in human cerebral spinal fluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysate fractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for total protein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. The FR results suggested that the surface modified catheters were less sensitive to the pressure and provide higher precision, and provided a FR closer to 100%. The surface modification did not show a significant effect on the protein EE. The average total protein EE of surface modified catheters was slightly higher than that of the native ones. The MS EE data of individual proteins showed a clear trend of complex response in EE with pressure.

Evaluation of a combined linear-nonlinear approach for column characterization using modern alkaline-stable columns as model.

This study investigates if deeper understanding is achieved when combining nonlinear and linear chromatographic column characterization methods. As test systems, two hybrid columns (Phenomenex Gemini-NX C18 and Kromasil Eternity C18) and one classic one (Kromasil-C18) were selected. The nonlinear methods were based on firm adsorption theory and involved determination of adsorption isotherms followed by calculations with a new numerical tool, adsorption energy distribution, on probe components at different pH values. The linear methods involved the hydrophobic subtraction model and selected probe components retention factors as a function of pH. The combined analysis indicated that both complementary and confirmative information can be achieved regarding the actual model systems.

MS for investigation of time-dependent protein adsorption on surfaces in complex biological samples.

AIM: This study aims at developing a nondestructive way for investigating protein adsorption on surfaces such as biomaterials using mass spectrometry. METHODS: Ventricular cerebrospinal fluid in contact with poly carbonate membranes were used as adsorption templates and on-surface enzymatic digestion was applied to desorb proteins and cleave them into peptides. Mass spectrometric analysis provided both protein identification and determination of protein specific adsorption behavior. RESULTS: In general, the adsorption increased with incubation time but also protein-specific time-resolved adsorption patterns from the complex protein solution were discovered. CONCLUSION: The method developed is a promising tool for the characterization of biofouling, which sometimes causes rejection and encapsulation of implants and can be used as complement to other surface analytical techniques.

Expanding the elution by characteristic point method for determination of various types of adsorption isotherms.

Important improvements have recently been made on the elution by characteristic point (ECP) method to increase the accuracy of the determined adsorption isotherms. However, the method has so far been limited/used for only type I adsorption isotherms (e.g. Langmuir, Tóth, bi-Langmuir). In this study, general strategies are developed to expand the ECP method for the determination of more complex adsorption isotherms including such containing inflection points. We will exemplify the methodology with type II, type III and type V isotherms. Guidelines are given for how to determine such isotherms using the ECP method and for the experimental considerations that must be taken into account or that may be eliminated in the particular case.

Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase.

An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate-adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.

Improvement in the generation of adsorption isotherm data in the elution by characteristic points method--the ECP-slope approach.

The elution by characteristic points (ECP) method is a very rapid and precise method for determination of the phase system equilibrium of phase systems in broad solute concentration ranges. Thus, the method is especially suitable for rapid characterization of high efficient separation systems. One important source of error, the effects by the post-loop dispersion, was eliminated in a recent investigation. In this study, the systematic error caused by the selection of the integration starting point at concentration equal to 0 is eliminated. This is done by developing and validating a new procedure for isotherm data generation; the ECP-slope method. The method generates raw slope data of the adsorption isotherm instead of raw adsorption data by integrations as the classical ECP does. Both numerical and experimental data were used for the comparison of the classical ECP approach with the slope-ECP method.