University of Debrecen

In this work, we present the synthesis and application of fluorescent rhodamine B hydrazide for the derivatization of simple oligosaccharides and complex glycans using a hydrazone formation chemistry approach. The labeling conditions and the experimental setup of CE/LIF were optimized by analyzing oligosaccharide standards. The CE/LIF separations were performed in polybrene-coated capillaries eliminating the need for the purification step after derivatization. The addition of methanol to the background electrolyte significantly increased the LIF detection sensitivity reaching the limits of detection in the attomole range. The resolution of carbohydrate samples was improved by using long (98 cm) capillaries and polymer additives (polybrene). The developed method was applied for CE/LIF and CE-MS analysis of N-linked glycans released from bovine ribonuclease B and the therapeutic monoclonal antibody of trastuzumab.

The Rare Gynecologic Sarcoma study involved 23 institutions from 10 countries focusing on myxoid leiomyosarcoma and non-smooth muscle uterine sarcomas. Here, we present the main results of the study, including the comparison between the original and final diagnosis, the frequency and type of molecular aberrations, and the clinicopathologic outcomes. A total of 379 cases were included, with available results for next-generation sequencing (NGS) RNA in 338 of 379 cases and NGS DNA in 335 of 379 cases. According to the original diagnoses, the study included 204 cases of low-grade endometrial stromal sarcoma (LG-ESS), 75 cases of high-grade endometrial stromal sarcoma (HG-ESS), 74 cases of undifferentiated uterine sarcoma (UUS), 17 cases of myxoid leiomyosarcoma, and 9 cases of unclassifiable sarcoma. The results of our second reading showed that 29% (110/379) of all the tumors had been originally misdiagnosed. After the reclassification, the final diagnoses were 147 cases of LG-ESS, 69 cases of HG-ESS, 58 cases of UUS, 3 cases of LG-ESS with high-grade transformation, 7 cases of perivascular epithelioid cell tumor, 9 cases of uterine tumor resembling ovarian sex cord tumor, 8 cases of tumors with a KAT6B/A::KANSL1 fusion, 2 cases of tumors with an NTRK fusion, 29 cases of undifferentiated carcinoma, and 47 tumors with smooth muscle differentiation. The molecular testing showed that LG-ESS harbor a recurrent fusion in 75.9% and HG-ESS in 43.7% of cases. The results of our study emphasize the diagnostic, prognostic, and predictive significance of molecular testing in mesenchymal uterine tumors.

Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) is a frequently used analytical technique in size-based separation of proteins, playing a vital role in the biopharmaceutical industry for the analysis and characterization of therapeutic proteins, employing both UV and fluorescent detection. Understanding the effect of the operational parameters using easily applicable in migratio fluorescent labeling is increasingly critical, especially because multicapillary electrophoresis systems with fluorescent detection have recently gained prominence in high-throughput biopolymer analysis. In this study, the effects of the three most important user-adjustable operational parameters (temperature, gel concentration, and electric field strength) were investigated on the electrophoretic mobility and resolution of SDS-protein complexes in the presence of propidium iodide in the gel-buffer system. In the separation temperature study, the Arrhenius equation was applied to calculate the required activation energy for the electromigration through the propidium iodide-containing separation matrix for the sample proteins. With increasing gel concentration, the presence of the fluorophore in the gel-buffer system resulted in linear Ferguson plots, suggesting more predictable and consistent sieving behavior, in contrast to the non-linear plots observed earlier without the addition of the fluorescent dye. Finally, increase in the applied electric field resulted in elevated electrophoretic mobilities, both in the presence and absence of propidium ions in the sieving matrix. The resolution between the consecutively migrating SDS-protein complexes, on the other hand, decreased above 500 V/cm, probably due to conformation changes caused by the high field strengths. Our results underline the importance of optimizing these key parameters in SDS-CGE with propidium-mediated LIF detection to obtain rapid and high-resolution separation of complex protein samples including biopharmaceuticals.