Project description:Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest especially due to the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in a case of very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields which were compared with the results from the same sample without precipitation. With this study it was possible to conclude that, in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery, number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone when comparing to the original sample.
Project description:In order to study the gene expression differences of ev76 in the environment and the appropriate growth temperature of flea (21 ℃) and mouse body temperature (37 ℃), we cultured ev76 at 21 ℃ and 37 ℃ respectively, collected bacterial precipitation and sequenced the transcriptome
Project description:In this study, institutional IRB approval was obtained and peripheral blood and bone marrow samples on patients with chronic myelomonocytic leukemia were collected. On the samples, we performed chromatin immuno-precipitation and next generation sequencing to assess histone modifications and epigenetic changes associated with proliferative and dysplastic phenotypes.
Project description:Limited proteome coverage is a common challenge in plasma or serum proteomic analysis. We find that sequential precipitation of plasma by increasing concentrations of acetonitrile yields distinct subsets of proteins. Deep analysis of the original whole plasma (Plasma), the pellets of plasma precipitated sequentially by 40% acetonitrile (40AcN_pellet) and further by 50% acetonitrile (50AcN_pellet), and the final supernatant (50AcN_Sup), have profiled 2040, 2078, 1153 and 1414 proteins respectively, yielding 3386 proteins in total. In addition, the plasma was also isolated by 10% and 20% PEG6000 precipitation followed by albumin depletion (10PEG/Alb-/-, 20PEG/Alb-/-) as we have previously reported (Anal Chem 2025, PMID: 40468192). Combination of these six samples (6mix), including: 1) the original plasma, 2) 20AcN_pellet (precipitate of plasma by 20% acetonitrile), 3) 40AcN_pellet, 4) 50AcN_Sup, 5) 10PEG/Alb-/-, and 6) 20PEG/Alb-/- has led to identification of 5441 proteins (5356 genes), increasing the proteome coverage remarkably. Plasma sample used for these analyses were pooled from 100 clinical samples (80 patients with different types of diseases + 20 visitors for health screening). Samples for the five in-depth proteomic datasets are: Plasma (the original whole plasma), 40AcN_pellet (the pellet of plasma precipitated by 40% acetonitrile), 50AcN_pellet (the pellet of the plasma was first precipitated by 40% acetonitrile and then by 50% acetonitrile), 50AcN_Sup (the final supernatant of plasma after precipitation by 50% acetonitrile), and 6mix (the equal mixture of the plasma and the five isolates as mentioned).