Epitope characterization of proteins and aptamers with mass spectrometry.

The way in which professor Michael Przybylski has combined the spirit of research with entrepreneurship has set an example for any and all scientists. He has made significant achievements in the fields of mass spectrometry, biochemistry and medicine, and has initiated important technological developments in the area of protein analysis. Between 2016 and 2023 professor Przybylski's scientific focus shifted on protein interactions with emphasis on aptamer-protein and antibody-protein analysis. This review focuses on professor Przybylski's achievements in the last few years highlighting his impact on the scientific community, on his students and colleagues.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination.

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry.

Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight α-helices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant KD of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146-153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a ß chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

Simplified measurement of middle molecular toxin removal during continuous therapies.

The removal of middle molecules during continuous extracorporeal detoxification therapies is a scientific field of increasing attention. However, it is difficult to find suitable and inexpensive markers and methods regarding the in vitro clearance of molecules in a weight range between 10 and 20 kDa. We present an easy and reliable cytochrome C-based photometric method to compare the in vitro clearance of different hemofilters during hemodialysis therapy. Cytochrome C is an inexpensive globular protein that shows a relative absorption maximum at λ = 410 nm, allowing photometric concentration assessment in the dialysate outflow line. Various hemofilters were evaluated to assess the removal of cytochrome C during simulated continuous venovenous hemodialysis. Although reported sieving coefficients for globular proteins and surface area of all hemofilters were similar, clearance differences of up to 55% were observable at blood and dialysate flows of 150 ml/min and 4,000 ml/h, respectively. We found that even in a strict hemodialysis setting with moderate blood flow, a remarkable diffusive cytochrome C clearance can be achieved. Hereby, diffusion is eventually supported by transmembrane flow and backflow in the proximal and distal sections of the hollow fiber of a hemofilter.

Treatment of severe hypercalcemia using continuous renal replacement therapy with regional citrate anticoagulation.

We report a patient with severe hypercalcemia and acute kidney failure, in whom citrate anticoagulation was used not only for anticoagulation but also to correct ionized hypercalcemia (1.77 mmol/L). In this patient, after a complicated surgical procedure, septic shock led to acute kidney failure. We started continuous venovenous hemodialysis with citrate anticoagulation. By almost stopping the calcium substitution during the first hours, elevated systemic ionized calcium decreased into the normal range within 8 hours. Although calcium substitution was then increased, serum ionized calcium decreased to a nadir of 0.86 mmol/L and then stabilized within the normal range within the next 24 hours. To correct the imbalance in systemic ionized calcium concentration, the calcium substitution was varied over a wide range of 0.1-3.0 mmol/L of generated effluent. The time delay between adjustment in calcium infusion rate and the first detectable change in ionized calcium level was below 4 hours. However, the full response to a change of the calcium substitution was found after 8-12 hours.

Assessment of temporary dialysis catheter performance on the basis of flow and pressure measurements in vivo and in vitro.

The efficiency of hemodialysis treatments depends on catheter performance and, consequently, on effective blood flow that can be achieved at maximum extracorporeal pressures. Differences in effective and displayed flow were determined with ultrasound dilution technology, and a mathematical correction function for the MultiFiltrate hemodialysis machine was developed. This algorithm was used to calculate effective blood flow during treatment from displayed flow and arterial pressure. To assess catheter performance over time, we measured effective blood flow as function of extracorporeal pressure in 11 uncuffed, tunneled hemodialysis catheters with shotgun design. Pressure and flow profiles of the catheters were determined, and pressure symmetry was measured. To assess flow resistance over time, pressure trends of the catheters at different blood flow rates were measured for each patient over a mean period of 6.1 +/- 3.0 days. Increases in flow resistance during the study period were found to be small. Mean arterial pressure decreased from -185 mm Hg to -200 mm Hg, and mean venous pressure increased from 197 mm Hg to 215 mm Hg. Effective flow did not change significantly during the study. In conclusion, all catheters investigated easily provided effective flows above 450 mL/min over the study period at maximum extracorporeal pressures below +/-300 mm Hg.

A new technique for establishing dry weight in hemodialysis patients via whole body bioimpedance.

BACKGROUND: Quantitative techniques are necessary to achieve dry weight (DW) in patients with kidney failure. Bioimpedance spectroscopy (BIS) is a non-invasive method that determines the volume of body fluid compartments. The current work evaluates the use of BIS data in hemodialysis patients for the prediction of DW. METHODS: A new technique has been devised for the estimation of DW that involves the intersection of two slopes, slope normovolemia (SNV) and slope hypervolemia (SHV). These slopes characterize the variation in extracellular water (ECW) with body weight (BW) in the states of normovolemia and hypervolemia, respectively. SNV was established via measurements of ECW and BW in 30 healthy subjects. In a longitudinal study in new hemodialysis patients, successive reduction of post-dialysis weight (PDW) was attempted until clinical signs of normovolemia were presented. Measurements of ECW and BW that were acquired at the beginning of each treatment were used to determine SHV. RESULTS: SNV was found to be 0.239 L/kg and 0.214 L/kg for male and female healthy subjects, respectively. A significant DeltaPDW predicted by the new method (-4.98 kg) was highly correlated to the DeltaPDW achieved in the study (-5.85 kg, R = 0.839). Blood pressure was reduced (P < 0.001) and an 86% decrease in antihypertensive agents was achieved. CONCLUSION: The method of intersecting slopes (SHV with SNV) via BIS is a new method for the prediction DW. This approach will offer considerable improvement for the routine management of DW in the dialysis setting.