Project description:Label free quantification (LFQ) and isobaric labelling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT 10-plex workflow to label free single shot data-independent acquisition (DIA) method on a controlled sample set. The sample set consisted of ten samples derived from 10 different mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. To match instrument time between the methods, the combined TMT sample was fractionated into ten fractions. The LC-MS data were acquired at two facilities to assess inter-laboratory reproducibility. Both methods resulted in a high proteome coverage (>5,000 proteins) with low missing values on protein level (<2%) The TMT workflow led to 15-20% more identified proteins and a slightly better quantitative precision whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA performed similar. The quantitative performance of the DIA data could be even improved by searching them directly against a database instead of using a project specific library. Our experiments also demonstrated that both methods can be easily transferred between facilities.
Project description:The lysosome, as the main degradative organelle of eukaryotic cells, is involved in numerous cellular processes. A defect in one of its proteins often results in lysosomal storage diseases (LSDs). For the study of lysosomal proteins, mass spectrometry (MS) has emerged as the method of choice. Lysosomal proteins are, however, low-abundant, restricting the analysis of lysosomal proteins in unbiased approaches to the investigation of lysosome-enriched fractions. The use of targeted MS provides an attractive alternative to analyze lysosomal proteins in a complex background. In this study, the two targeted MS approaches data-independent acquisition (DIA) and parallel reaction monitoring (PRM) were compared with regard to their ability to analyse lysosomal proteins. These experiments were conducted with samples of different complexity: low-complex lysosome-enriched fractions, medium-complex mouse embryonic fibroblasts (MEF), and high-complex liver whole tissue lysate. While both MS approaches were able to identify and quantify lysosomal proteins, PRM outperformed DIA, especially in high-complex samples.
Project description:Obesity and type 2 diabetes (T2D) remain major global healthcare challenges and developing therapeutics necessitate using nonhuman primate models. Here, we present proteomic analyses of all the major organs of cynomolgus monkeys with spontaneous obesity or T2D in comparison to healthy controls.
Project description:In the present study we used DIA MS to characterize the profile of over 4,000 proteins in immortalized human astrocytes exposed to TNF, IL-1β, and LPS, as well as in primary human astrocytes cocultured with brain endothelial cells and exposed to LPS. For both immortalized and primary astrocytes, DIA MS data were matched against a spectral library generated by merging DDA MS runs from both unfractionated and fractionated cell lysates.
Project description:Human African trypanosomiasis (HAT) caused by the extracellular protozoon Trypanosoma brucei, is a neglected tropical disease affecting the poorest communities in sub-Saharan Africa. HAT progresses from a hemolymphatic first stage (S1) to a meningo-encephalitic late stage (S2) when parasites reach the central nervous system, although the existence of an intermediate stage (Int.) has also been proposed. The pathophysiological mechanisms associated with the development of S2 encephalopathy are yet to be fully elucidated. In the present study, we used data independent acquisition mass spectrometry (DIA-MS) to investigate the potential pathogenic effects of microvesicles (MVs) derived from HAT patients’ cerebrospinal fluid (CSF). To assess potential biological properties of these MVs, immortalized human astrocytes were exposed, in vitro, to MVs enriched from S1, Int. or S2 CSF. DIA-MS analyses showed that S2 MVs induced, compared to Int. or S1 MVs, a stronger proteome modulation in astrocytes that resembled the one produced by IFN-γ, a key molecule in HAT pathogenesis. Our results indicate that HAT S2 CSF harbors MVs potentially involved in the mechanisms of pathology associated with HAT late stage. Such vesicles might thus represent a new player to consider in future functional studies.
Project description:In this project, two NSCLC cohorts were analyzed by Data Independent Acquisition (DIA-MS). A cohort of early stage NSCLC samples (141) as well as an additional cohort of late stage NSCLC samples (84) with the aim to demonstrate utility of MS for subtyping and treatment prediction in a clinical setting. Further, six identified NSCLC proteome subtypes were investigated in relation to cancer driver pathways and immune phenotypes based on the generated MS-data.
Project description:In this project, one NSCLC cohorts were analyzed by Data Independent Acquisition (DIA-MS). A cohort of early stage NSCLC samples (207) with the aim to demonstrate utility of MS for subtyping and treatment prediction in a clinical setting. Further, six identified NSCLC proteome subtypes were investigated in relation to cancer driver pathways and immune phenotypes based on the generated MS-data.
Project description:Here, we developed immunoprecipitation-mass spectrometry assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells.
Project description:Tremella fuciformis is a dimorphic fungus that can undertake the reversible transition between yeast-like spores and hypha forms. In this project, we attempted to explore the differential proteins profile of dikaryotic yeast-like spores (FBMDS) and dikaryotic mycelium (DM) by applying the HRMS1-DIA full proteomics and PRM target proteomics synthetically. The results showed that a total of 5687 proteins were quantified, and 2220 of them (39.01%) showed more than a two-fold change in expression. The functional analysis of differential expression proteins (DEPs) confirmed that the DEPs were mainly located on membrane and nucleus, and FBMDS tended to express proteins involved in DNA replication and transcription, DNA damage repair, biosynthesis, and metabolism. At the same time, DM exhibited increased expression of signal transduction such as MAPK signaling pathway, Ras signaling pathway.
Project description:Quantitative proteomics in DIA mode was used to analysis the proteomic profile of 24 weeks from the sorafenib treated and vehicle treated monkeys.