Series of Concentration-Induced Phase Transitions in Cholesterol/Phosphatidylcholine Mixtures

In lipid membranes, temperature-induced transition from gel-to-fluid phase increases the lateral diffusion of the lipid molecules by three orders of magnitude. In cell membranes, a similar phase change may trigger the communication between the membrane components. Here concentration-induced phase transition properties of our recently developed statistical mechanical model of cholesterol/phospholipid mixtures are investigated. A slight (<1%) decrease in the model parameter values, controlling the lateral interaction energies, reveals the existence of a series of first- or second-order phase transitions. By weakening the lateral interactions first, the proportion of the ordered (i.e., superlattice) phase (Areg) is slightly and continuously decreasing at every cholesterol mole fraction. Then sudden decreases in Areg appear at the 0.18–0.26 range of cholesterol mole fractions. We point out that the sudden changes in Areg represent first- or second-order concentration-induced phase transitions from fluid to superlattice and from superlattice to fluid phase. Sudden changes like these were detected in our previous experiments at 0.2, 0.222, and 0.25 sterol mole fractions in ergosterol/DMPC mixtures. By further decreasing the lateral interactions, the fluid phase will dominate throughout the 0.18–0.26 interval, whereas outside this interval sudden increases in Areg may appear. Lipid composition-induced phase transitions as specified here should have far more important biological implications than temperature- or pressure-induced phase transitions. This is the case because temperature and pressure in cell membranes are largely invariant under physiological conditions

Inter- and intratumoral proteomics and glycosaminoglycan characterization of ALK rearranged lung adenocarcinoma tissues: a pilot study

Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation-reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions

Self-regulating genes. Exact steady state solution by using Poisson representation

Systems biology studies the structure and behavior of complex gene regulatory networks. One of its aims is to develop a quantitative understanding of the modular components that constitute such networks. The self-regulating gene is a type of auto regulatory genetic modules which appears in over 40% of known transcription factors in E. coli. In this work, using the technique of Poisson Representation, we are able to provide exact steady state solutions for this feedback model. By using the methods of synthetic biology (P.E.M. Purnick and Weiss, R., Nature Reviews, Molecular Cell Biology, 2009, 10: 410-422) one can build the system itself from modules like this.Comment: 10 pages, 2 figures, 1 table, 1 supplemental material (9 pages); additional reference to the work of Grima et a

Synovialis folyadékból nyert extracellularis vesiculák proteomja és azok lehetséges funkciója befertőződött ízületi protézisekben

Bevezetés: A periprotetikus infekciók ellátása jelentős kihívás elé állítja az operáló orvost, mind diagnosztikai, mind terápiás tekintetben. Az utóbbi években a mozgásszervi kutatások során egyre növekvő figyelmet kaptak az extracellularis vesiculák. Az extracellularis vesiculák által szállított fehérjék tömegspektrometrián alapuló azonosítása fontos lépés, mely segíthet megérteni a védekezési folyamatban betöltött biológiai funkcióikat. Célkitűzés: Vizsgálatunk célja volt az akut és a krónikus fertőzött mintákból izolált extracellularis vesiculák fehérjetartalmának megismerése, azonosságok és különbségek keresése – az „egy legjobb biomarker” megtalálása helyett a lehető legtöbb, detektálható mennyiségben jelen lévő extracellularis vesiculába zárt fehérje vizsgálata és biológiai folyamatokba illesztése. Módszer: Prospektív, monocentrikus vizsgálatot végeztünk, a beválasztási kritériumok a 2018-as MSIS-kritériumokon alapultak. A vizsgálatba 13 (n = 13) beteget vontunk be, minden beteg periprotetikus infekció miatt került műtétre. 6 (n = 6) betegnél akut purulens (akut csoport) folyamatot, míg 7 (n = 7) betegnél ’low-grade’ infekciót (krónikus csoport) igazoltunk. Az extracellularis vesiculák izolálása minden esetben a protézist körülvevő synovialis folyadékból történt. A tömegspektrometriai vizsgálattal azonosított fehérjék funkcionális alapú klaszterezésére a STRING, KEGG, Gene Ontology adatbázisokat használtuk. A végleges vizualizáció Cytoscape 3.9.1. szoftverrel történt. Eredmények: Az extracellularis vesiculák feltárása után 222 db fehérjét azonosítottunk, melyek vagy az akut, vagy a krónikus minták valamelyikének több mint felében fordultak elő. Csak az akut minták több mint felében 50 db fehérjét; csak a krónikus minták több mint felében 33 db fehérjét; egyszerre mindkét csoport több mint felében 86 db fehérjét azonosítottunk. Ezek alapján készültek a funkcionális klaszterek. Megbeszélés: A protézisfertőzések diagnosztikájában régóta megvan a törekvés, hogy megtalálják az „egy legjobb biomarkert”, amely biztosan különbséget tud tenni fertőzött és nem fertőzött protézislazulás között. Következtetés: Vizsgálatunk célja nem egy újabb biomarker kiválasztása volt, hanem az extracellularis vesiculákban szállított fehérjék biológiai folyamatokban betöltött szerepének ábrázolása, leírása, amellyel jobban betekinthetünk a periprotetikus infekció során zajló folyamatokba. Orv Hetil. 2024; 165(3): 98–109

Salt gradient chromatographic separation of chondroitin sulfate disaccharides

In the present study, we describe the development of a fast, 2-step salt gradient for analysis of chondroitin sulfate disaccharides. Using salt gradients, which is somewhat unusual in HILIC-based separations, provides relatively fast chromatography with excellent sensitivity (15 min cycle time, 10–20 fmol/µL detection, 30–50 fmol/µL quantitation limit), and good linearity. The efficiency of the new method is demonstrated by measuring human tissue slices of healthy, cirrhotic, and cancerous liver samples. Preliminary results show major differences among the quantity and sulfation pattern of the various sample types

Proteomics and N-glycosylation analysis of prostate cancer biopsies

Introduction Analysis of tissue specimen using mass spectrometry offers valuable information of biological processes taking place at the origin of a disease. Identifying differences in protein expression levels and site-specific N-glycosylation of glycoproteins among cancerous and healthy tissues can be an attractive approach in biomarker research. Our aim was to analyze prostate cancer (PCa) tissue microarray samples (n=95) and compare protein expression levels and changes in N-glycosylation features among various pathological grades of PCa and healthy tissues. Methods Following on surface proteolytic digestion [1] glycopeptides were enriched using acetone precipitation. Peptide and glycopeptide fractions were analyzed separately using reversed phase nanoHPLC-MS(MS) and nanoHPLC-MS. Label free protein quantitation was performed using MaxQuant, while glycopeptides were identified and quantified using Byonic and Glycopattern software, respectively. Statistical analysis was carried out using Perseus. Results Several statistically significant proteins were identified that were altered between different cancer grades and healthy tissue. STRING analysis revealed protein pathways involved in disease progression. Regarding N-glycosylation analysis the changes identified at specific glycosylation sites were of interest. Alterations in sialylation, fucosylation and galactosylation of individual glycosites were detected that could potentially be important targets of future studies. We found that in case of Collagen 6 subunit both protein expression levels and fucosylation at N785 changed significantly between the different pathological grades and healthy tissue. Conclusions Our integrated proteomics and glycoproteomics workflow was used to identify molecular alterations occurring during PCa progression from limited size tissue samples and can also be used to analyze other types of cancerous tissue biopsies in the future

GLYCOPEPTIDE ENRICHMENT USING SOLVENT PRECIPITATION

Glycoproteins have essential roles in biology and medicine. Glycosylation patterns often change during pathophysiological conditions which is also underlined by the fact that most tumor biomarkers are glycoproteins. Biopsies are generally stored in the form of tissue microarrays (TMA) facilitating parallel analysis of healthy, cancerous and metastatic tissues. The aim of our work is to characterize the glycosylation pattern of prostate cancer TMAs following on-surface tryptic digestion. For sensitive detection of minor glycoforms in tissues, especially that using very small sample amounts like that available in TMAs, it is essential to include a glycopeptide enrichment step in the analytical workflow. First we tested a mixed mode graphite and C18 SPE column, but this did not prove efficient in binding glycopeptides. Subsequently we used a solvent (acetone) based precipitation method1, which gave promising results. Following initial trials, we have optimized the method to achieve both high yield and high enrichment for glycopeptides. Modified parameters included the pH, salt concentration, the volume and the type of the precipitating solvent. Results were tested both on a model glycoprotein, alpha-1-acid glycoprotein (AGP) and also on a complex HeLa cell lysate. Glycopeptides and peptides were identified by tandem mass spectrometry in a nano-LC-MS/MS workflow using the Byonic software. Quantitation of the individual glycoforms was performed based on MS1 using our in-house GlycoPattern software. Results of the optimization and glycopeptide enrichment will be presented

Alterations in protein expression and site-specific N-glycosylation of prostate cancer tissues

Abstract Identifying molecular alterations occurring during cancer progression is essential for a deeper understanding of the underlying biological processes. Here we have analyzed cancerous and healthy prostate biopsies using nanoLC-MS(MS) to detect proteins with altered expression and N-glycosylation. We have identified 75 proteins with significantly changing expression during disease progression. The biological processes involved were assigned based on protein–protein interaction networks. These include cellular component organization, metabolic and localization processes. Multiple glycoproteins were identified with aberrant glycosylation in prostate cancer, where differences in glycosite-specific sialylation, fucosylation, and galactosylation were the most substantial. Many of the glycoproteins with altered N-glycosylation were extracellular matrix constituents, and are heavily involved in the establishment of the tumor microenvironment

Selective TiO2 phosphopeptide enrichment of complex samples in the nanogram range

Phosphopeptide enrichment is a commonly used sample preparation step for investigating phosphorylation. TiO2-based enrichment has been demonstrated to have excellent performance both for large amounts of complex and for small amounts of simple samples. However, it has not yet been studied for complex samples in the nanogram range. Our objective was to develop a methodology applicable for complex samples in the low nanogram range, useful for mass spectrometry analysis of tissue microarrays. The selectivity and performance of two stationary phases (TiO2 nanoparticle-coated monolithic column and spin tip filled with TiO2 microspheres) and several loading solvents were studied. Based on this study, we developed an effective and robust method, based on a spin tip with a non-conventional 50 mM citric acid-based loading solvent. It gave excellent results for phosphopeptide enrichment from samples containing a few nanograms of a complex protein mixture. © 2020 by the authors. Licensee MDPI, Basel, Switzerland