| 1. |
- Nezhyva, Mariya, et al.
(författare)
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Inquiry-based Learning of Proteomics and Metabolomics
- 2024
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Ingår i: Journal of Chemical Education. - : American Chemical Society (ACS). - 0021-9584 .- 1938-1328. ; 101:2, s. 521-529
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Tidskriftsartikel (refereegranskat)abstract
- Given the rapid development of data-driven experimental procedures within the life sciences, it is important to equip students with proper skills and knowledge on how to obtain and interpret complex data. While laboratory exercises have for a long time been well established as a teaching method for life sciences, we consider there is room for improvement in how laboratory exercises are conducted. Hence, we designed a laboratory exercise course in which students at the graduate level in the European education system (M.Sc.) are challenged to pose their own biological question and write their own laboratory protocol for a proteomic study to investigate their hypothesis. Here, students are supported in their task with lectures and seminars that take them through the required details on experimental sample preparation, analysis with LC-MS, and proteomic data evaluation of biological function and relevance. According to student interviews, the inquiry-based learning concept we used here provided a deeper understanding of the laboratory protocols they wrote, according to which they eventually performed their own experiments.
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| 2. |
- Nezhyva, Mariya
(författare)
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Integrative Approaches in Shotgun Proteomics : From sample preparation to multifaceted data analysis
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bottom-up proteomics mass-spectrometry gives an opportunity to shed light on var-ious biological aspects/characteristics such as protein composition, its modifications,interactions, and dynamics. Its applications span a wide range of biological sciencesaiming to eventually answer fundamental disease related questions or evaluate drugperformance and predict adverse effects.Bottom-up proteomics experiments are worth the time and effort because it is a comprehensive and multi-level approach to address research questions from a single dataset. Hence, we can gain a thorough understanding of the processes in the studied system and their interconnected changes, thereby confirming results from different perspectives. While the majority of research is focused on the improvement of analytical techniques, it remains challenging to derive meaningful biological insights from the data.This Ph.D. thesis contributes to various proteomics challenges, including the development of a high-throughput micro-scale proteomic workflow and comprehensive multifaceted analysis. In particular, the data on the thermal proteome profiling of melanocyte-stimulating hormone interactions with melanocortin receptors was analyzed using a newly developed workflow. This workflow uniquely combines thermal stabilization data, inferred transcription factor activity, and pathway analysis. Additionally, this thesis integrates omics approaches such as metabolomics and proteomics. Another dimension to the dataset was added by combining metabolic mass spectrometry imaging with region-specific proteomics for the characterization of cocaine’s neurotoxicity.
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| 3. |
- Nezhyva, Mariya, et al.
(författare)
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POMC-specific Modulation of Metabolic and Immune Pathways via Melanocortin-3 Receptor Signaling
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- This proteomic study provides a nuanced understanding of the melanocortin-3 receptor (MC3R) and its ligand-specific effects on metabolic and immune pathways. Utilizing thermal proteome profiling with tandem mass spectrometry, we uncovered the distinct influences of adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and γ-melanocyte-stimulating hormone (γ-MSH) on protein thermal stability and pathway activation. ACTH uniquely affected NADPH-related metabolic proteins, α-MSH significantly modulated the IL-6 pathway via STAT3, and γ-MSH prominently activated interferon signaling. All ligands shared involvement in the cAMP-PKA-CREB and varied impacts on PI3K and ERK pathways, crucial for energy metabolism. Additionally, ligand-specific responses in mitochondrial protein stability suggest a role in cellular energy generation.
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| 4. |
- Nezhyva, Mariya, et al.
(författare)
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Spatial multiomic insights into acute cocaine exposure
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Recent studies provide compelling evidence that cocaine-induced neurotoxicity begins within hours of a single acute cocaine exposure. Despite this, a comprehensive understanding of the molecular alterations occurring in vivo within the reward system following such an exposure has been lacking. In this study, we developed an analytical workflow that combines mass spectrometry imaging with microscale proteomics of brain regions. Here, we present a multiomic perspective on the molecular consequences of acute cocaine exposure on the principal areas of the reward system and the hippocampus. Our findings include distinct region-specific alterations in the tricarboxylic acid (TCA) cycle and lipid synthesis within the reward circuitry highlighting a significant energy depletion in mice 24 hours post-cocaine injections. Additionally, we linked widespread reductions in key neurotransmitters (GABA, glutamate, aspartate) across the reward system and calcium level modifications to changes in synaptic plasticity and mitochondria dysfunction. Mitochondrial dysfunction and energy metabolism disruption were evident through imbalances in the mitochondrial ATP production and electron transport chain components, increased susceptibility to oxidative stress, disturbances in mitochondrial transport proteins, and fluctuations in creatine and taurine levels. Among the brain regions within the reward circuitry, the prefrontal cortex (PFC) exhibited the most pronounced effects. This study not only provides a holistic overview of the intricate interplay between proteins and metabolites within the reward circuitry regions during the onset of cocaine-induced neurotoxicity but also offers novel insights into the underlying molecular mechanisms.
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| 5. |
- Sandbaumhuter, Friederike A., et al.
(författare)
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Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:41, s. 15400-15408
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Tidskriftsartikel (refereegranskat)abstract
- Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC-MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC-MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, alpha-MSH, and gamma-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC-MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.
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| 6. |
- Sandbaumhüter, Friederike A., et al.
(författare)
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Well-Plate muFASP for Proteomic Analysis of Single Pancreatic Islets
- 2022
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 21:4, s. 1167-1174
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Tidskriftsartikel (refereegranskat)abstract
- Filter-aided sample preparation (FASP) is widely used in bottom-upproteomics for tryptic digestion. However, the sample recovery yield of this methodis limited by the amount of the starting material. While similar to 100 ng of digested protein issufficient for thorough protein identification, proteomic information gets lost with aprotein content <10 mu g due to incomplete peptide recovery from thefilter. Wedeveloped and optimized aflexible well-plate mu FASP device and protocol that issuitable for an similar to 1 mu g protein sample. In 1 mu g of HeLa digest, we identified 1295 +/- 10proteins with mu FASP followed by analysis with liquid chromatography-massspectrometry. In contrast, only 524 +/- 5 proteins were identified with the standardFASP protocol, while 1395 +/- 4 proteins were identified in 20 mu g after standard FASPas a benchmark. Furthermore, we conducted a combined peptidomic and proteomicstudy of single pancreatic islets with well-plate mu FASP. Here, we separated neuropeptides and digested the remaining on-filterproteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved inglucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a usefultool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI:10.6019/PXD029039
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