Concatemer Assisted Stoichiometry Analysis (CASA) of Cell-free Reconstituted Kinetochores
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ABSTRACT: Concatemer-assisted stoichiometry analysis (CASA) addresses a common challenge in cell biology: absolute quantification of protein subunits in native protein complexes.
Project description:Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.
Project description:In this study, we systematically assessed the fine-tuning of limited proteolysis in the extracellular space by glycosylation, one of the most widely observed PTMs, using cells, and applying the Terminal Amine Labeling of Substrates (TAILS) approach, a quantitative proteomics workflow for the system-wide assessment of protein N termini and protease cleavage events in complex biological samples. In addtion, we complement the study with DIA analysis, PRM and glycoproteomics analysis for further validation of the initial observations
Project description:This study represents the first quantitative analysis of the temporal changes in the small urinary extracellular vesicle proteome throughout living donor kidney transplantation identifying PCK2 abundance as a biomarker for renal function 12 months after transplantation
Project description:Arabidopsis thaliana plant expressing 35S:WIND1 shows callus-like morphology without hormone treatment. Transcriptomes of the callus-like cell expressing 35S:WIND1, callus of T87 cultured cell, 2,4-D-induced callus and control seedling plant were compared by Agilent microarray. Comparison of four kinds of Arabidopsis thaliana plants. Biological replicates: three for each.
Project description:au11-02_cho-thf - shoots and roots treated with methotrexate and 5-formyl-thf - Folates and 1C metabolism - The objective of this study is to investigate the changes on the plant expression profiles due to 5-CHO-THF assimilation and transformation. Additionally, plants supplemented with methotrexate (MTX) (an inhibitor of folate biosynthesis) will complement the study. Results from this work will serve to better understand the effects of folate accumulation in plants. 12 dye-swap - treated vs untreated comparison
Project description:fapesp-bra-inra-10-01_bioen_hypocotyl - dark hypocotyls tor rnai - Transcriptional comparison between 2 TOR RNAi mutants versus GUS control. - Sowing after 24h imbibition at 4M-BM-0C in the dark, on MS1/5, no sucrose, 10 mM ethanol, 8 g/l agar, vertical growth with 3h light, 6 days growth in the dark (20M-BM-0C), hypocotyls were harvested under green light ; cotyledons and root were removed. 4 dye-swap - normal vs rnai mutant comparaison
Project description:VOZ1 and VOZ2 are recently identified highly unique transcription factors found in vascular plants and moss Physcomitrella patens. We isolated a voz1 voz2 double mutant of Arabidopsis thaliana. To determine the biological roles of VOZ1 and VOZ2, we perfomed a microarray experiment. voz1 voz2 double mutant and wild-type plants were grown on MS agar medium, and the transcriptomes of 14 day whole seedlings were compared. 4 replicates per genotype.
Project description:RNAseq of wild type Col-0 Arabidopsis plants and piezo/feronia/camta3 mutants before and 22 minutes after touch treatment with a gentle paint brush. The mutants were defective in genes involved in mechanosensing and downstream signalling. Their contribution to touch-responsive signalling was assessed by transcriptome analysis.