Characterization of the binding of apolipophorin III to lipopolysaccharides

Apolipophorin III (apoLp-III) is a small insect apolipoprotein used as a model. Apolipoproteins neutralize lipopolysaccharides but the details are poorly understood. Lipopolysaccharides of gram negative bacteria can induce a septic shock, a common lethal condition. In the present study, the apoLp-III binding interaction with lipopolysaccharides from a variety of bacterial species was investigated using FPLC, dynamic light scatter, tyrosine fluorescence and native PAGE. ApoLp-III binding to Escherichia coli lipopolysaccharides resulted in the formation of complexes with a molecular mass of 400 kDa, a diameter of 18.7 nm, with 4 apoLp-III and 24 LPS molecules. The complexes with Klebsiella pneumoniae LPS were somewhat smaller. Analysis with truncated and detoxified lipopolysaccharides indicated that apoLp-III preferably binds to lipid A, while the O-antigen repeat elicited a weaker binding interaction. These findings led to a new model of apolipoprotein-lipopolysaccharide interaction and may be used for development of new therapeutic approaches to treat sepsis

Bioactive Peptide Profiling in Collagen Hydrolysates: Comparative Analysis Using Targeted and Untargeted Liquid Chromatography–Tandem Mass Spectrometry Quantification

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides—Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)—in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699

Development and validation of an HPLC-UV method for purity determination of DNA

Determination of the concentration and the purity of DNAs is crucial for measuring the DNA copy number since it will influence further DNA analysis such as digital PCR (dPCR) and next-generation sequencing (NGS). Precise and scientifically validated DNA measurements empower healthcare professionals and authorities to deliver reliable outcomes. DNA agarose gel electrophoresis is commonly used to check DNA purity; however, its resolution is limited. In this study, a quantitative HPLC-UV measurement method to separate DNAs was established as an alternative to both DNA electrophoresis and spectrophotometric techniques. The method was fully validated to separate DNAs ranging between 75 and 20,000 base pairs. DNA mixtures were prepared gravimetrically. Chromatographic separations were conducted on a TSKgel DNA-NPR column with dimensions of 2.5µm, 4.6mm ID x 7.5cm, using a flow rate of 0.75 mL/min. The uncertainty of the method was assessed following the guidelines provided by EURACHEM/CITAC. The method demonstrated linearity for the 200 bp DNA fragment within the range of 0.4 ng to 800 ng DNA, with a high regression coefficient of R²=0.999. The Limit of Detection (LOD) for the 200 bp DNA fragment was determined to be 1 ng, while the Limit of Quantitation (LOQ) was found to be 3 ng. The recovery percentages for the 1%, 5%, and 10% impurities of the 150 bp DNA in 200 bp DNA fragments were measured at 101.8%, 97.4%, and 99.5%, respectively. The method established can be used in the value assignment stages of the reference materials which are required for SI traceable DNA measurements. [Med-Science 2023; 12(3.000): 876-82

Characterization of the ApoLp-III/LPS Complex: Insight into the Mode of Binding Interaction

Apolipoproteins are able to associate with lipopolysaccharides (LPS), potentially providing protection against septic shock. To gain insight into the molecular details of this binding interaction, apolipophorin III (apoLp-III) from <i>Galleria mellonella</i> was used as a model. The binding of apoLp-III to LPS was optimal around 37–40 °C, close to the LPS phase transition temperature. ApoLp-III formed complexes with LPS from <i>E. coli</i> (serotype O55:B5) with a diameter of ∼20 nm and a molecular weight of ∼390 kDa, containing four molecules of apoLp-III and 24 molecules of LPS. The LPS-bound form of the protein was substantially more resistant to guanidine-induced denaturation compared to unbound protein. The denaturation profile displayed a multiphase character with a steep drop in secondary structure between 0 and 1 M guanidine-HCl and a slower decrease above 1 M guanidine-HCl. In contrast, apoLp-III bound to detoxified LPS was only slightly more resistant to guanidine-HCl induced denaturation compared to unbound protein. Analysis of size-exclusion FPLC elution profiles of mixtures of apoLp-III with LPS or detoxified LPS indicated a much weaker binding interaction with detoxified LPS compared to intact LPS. These results indicate that apoLp-III initially interacts with exposed carbohydrate regions, but that the lipid A region is required for a more stable LPS binding interaction

Proteomic Analysis of Kidney Preservation Solutions Prior to Renal Transplantation

One of the main issues in kidney transplantation is the optimal functional preservation of the organ until its transplantation into the appropriate recipient. Despite intensive efforts, the functional preservation period remains limited to hours. During this time, as a result of cellular injury, various proteins, peptides, and other molecules are released by the organ into the preservation medium. In this study, we used proteomic techniques to analyze the protein profiles of preservation solutions in which organs had been preserved prior to their transplantation. Samples were obtained from the preservation solutions of 25 deceased donor kidneys scheduled for transplantation. The protein profiles of the solutions were analyzed using 2D gel electrophoresis/MALDI-TOF and LC-MS/MS. We identified and quantified 206 proteins and peptides belonging to 139 different groups. Of these, 111 proteins groups were belonging to kidney tissues. This study used proteomic techniques to analyze the protein profiles of organ preservation solutions. These findings will contribute to the development of improved preservation solutions to effectively protect organs for transplantation

Proteomic fertility markers in ram sperm

Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity

Proteomic Analysis of Liver Preservation Solutions Prior to Liver Transplantation

Objective: Transplantation is the preferred treatment for patients with end-stage liver diseases. However, in clinical practice, functional preservation of the liver is a major concern before the transplantation. Although various protective solutions are used (in combination with hypothermia), the functional preservation time for liver is still limited to hours. We analyzed the preservation medium to detect the proteins released from the liver during storage period