Percorrer por autor "Giddey, Alexander D."
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- Impact of PPP1R1A Knockdown on the Proteomic Landscape of INS-1 Cells: A Focus on Significant Modulated PathwaysPublication . Taneera, Jalal; Giddey, Alexander D.; Soares, Nelson C.; Khalique, Anila; Mohammed, Abdul Khader; Mahgoub, Mohamed Omer; Mahgoub, EglalPPP1R1A (protein phosphatase 1 regulatory inhibitor subunit 1A) is a cAMP/PKA-responsive inhibitor of protein phosphatase 1 (PP1) with a pivotal role in pancreatic β-cell physiology. To investigate its functional impact, Ppp1r1a was silenced in INS-1 (832/13) rat β-cells, and proteomic alterations were profiled using label-free DIA mass spectrometry (Orbitrap Exploris 480) with a rat spectral library. Quantitative analysis (n = 4/group) identified ∼2846 proteins with >2-fold change, revealing extensive proteome reprogramming. Key biological processes affected included vesicle trafficking and exocytosis, insulin biosynthesis and processing, organelle organization, mRNA processing, and autophagy. Pathway enrichment highlighted disruptions in insulin secretion, insulin resistance, and mTOR signaling. Crucial β-cell proteins, including INS2, Cacna1a, CPEB2, PCSK2, SNAP25, SYT5, and VAMP7, were significantly downregulated. Validation confirmed reduced phosphorylated AKT levels and p-AKT/T-AKT ratio, consistent with impaired mTOR signaling. Collectively, these findings demonstrate that PPP1R1A is essential for maintaining β-cell function and insulin secretion, and its depletion triggers broad proteomic and signaling alterations. Thus, PPP1R1A emerges as a regulatory node with potential therapeutic relevance in modulating β-cell activity and insulin dynamics in diabetes.
- Medwakh smoking induces alterations in salivary proteins and cytokine expression: a clinical exploratory proteomics investigationPublication . Aghila Rani, K.G.; Soares, Nelson C.; Rahman, Betul; Giddey, Alexander D.; Al-Hroub, Hamza M.; Semreen, Mohammad H.; Al Kawas, SausanBackground: Medwakh smoking has radically expanded among youth in the Middle East and around the world. The rising popularity of medwakh/dokha usage is linked to the onset of several chronic illnesses including cardiovascular diseases and cancers. Medwakh smoking is reported to increase the risk of inflammation in the lower respiratory tract owing to oxidative burden. To date, there are no reported studies investigating the impact of medwakh smoking on salivary protein profile. The current study aims to elucidate alterations in the salivary proteome profile of medwakh smokers. Methods: Saliva samples collected from 33 medwakh smokers and 30 non-smokers were subjected to proteomic analysis using UHPLC-ESI-QTOF-MS. Saliva samples were further subjected to validatory experiments involving analysis of inflammatory cytokine profile using LEGENDplex™ Human Essential Immune Response Panel. Results: Statistical analysis revealed alterations in the abundance of 74 key proteins including immune mediators and inflammatory markers in medwakh smokers (Accession: PXD045901). Proteins involved in building oxidative stress, alterations in cell anchorage, and cell metabolic processes were enhanced in medwakh smokers. Salivary immune response evaluation further validated the proteome findings, revealing significantly higher levels of IL-1β, IL-12p70, IL-23, IFN-γ (Th1 cytokines), IL-6 (Th2 cytokine), and MCP-1 (chemokine) in medwakh smokers. In addition, a substantial increase in abundance of involucrin suggesting a plausible stratified squamous cell differentiation and increased cell lysis in the oral cavity of medwakh smokers akin to chronic obstructive pulmonary diseases (COPD). The protein-metabolite joint pathway analysis further showed significantly enriched differentially expressed proteins and metabolites of glycolysis/gluconeogenesis, pentose phosphate, fructose and mannose, nicotinate and nicotinamide, and glutathione metabolism pathways among medwakh smokers. Conclusions: The findings of the study provide valuable insights on potential perturbations in various key immune molecules, cytokines, and signaling pathways among medwakh smokers. Medwakh smokers displayed elevated inflammation, increased oxidative stress and defective antioxidant responses, dysregulated energy metabolism, and alterations in proteins related to cell adhesion, migration, differentiation, and proliferation. The findings of study underscore the urgent need for comprehensive public health interventions among youth by raising awareness, implementing effective smoking cessation programs, and promoting healthy lifestyle to safeguard the well-being of individuals and communities worldwide.
- Optimizing MS Parameters for Data-Independent Acquisition (DIA) to Enhance Untargeted MetabolomicsPublication . Pinto, Frederico G.; Giddey, Alexander D.; Almarri, Rouda S. B.; Alkhnbashi, Omer S.; Garrett, Timothy J.; Uddin, Mohammed J.; Soares, Nelson C.Data-Independent Acquisition (DIA) has emerged as a powerful mass spectrometry (MS) strategy for comprehensive metabolomics. This study presents a novel short gradient (13 min) nanosensitive analytical method for human plasma analysis using DIA LC-MS/MS, focusing on in-depth optimization of MS parameters to maximize data quality and metabolite coverage. Key MS parameters, including scan speed, isolation window width, resolution, automatic gain control, and collision energy, were systematically tuned to balance the sensitivity and specificity while minimizing interferences. The optimized method enabled the detection of 2,907 features with 675 annotated compounds, leveraging recent progress in nano-LC-MS/MS for multiomics applications and showcasing the possibility of combining proteomics and metabolomics within a single chromatographic system. Ultimately, a comparison was performed between the data acquired through the DIA and DDA MS approaches in the context of untargeted metabolomics. This optimized analytical method yields more robust and reproducible results, thereby expanding the potential for meaningful discoveries across diverse biological fields.
- Repurposing proteomic nanoLC-MS platforms for untargeted metabolomics: evaluating DIA and polarity switching performance in human plasmaPublication . Pinto, Frederico G.; Giddey, Alexander D.; Mohamed, Nesrin; Almarri, Rauda S. B.; Murtaza, Munazza; Nassir, Nasna; Alkhnbashi, Omer S.; Uddin, Mohammed J.; Soares, Nelson C.Background: Many of the advanced MS methods applied in proteomics such as nanoflow LC-MS with data-independent acquisition have yet to be verified and/or optimized on metabolomics applications. Research design and methods: This study evaluates the feasibility of repurposing a proteomics-optimized nanoLC-MS platform for untargeted metabolomics. Using NIST SRM 1950 reference human plasma, we compared the performance of polarity switching and separate polarity modes under DIA conditions, focusing on metabolite coverage, annotation, and response linearity. Results: We observed, in the separate polarity and switching polarity runs 669 and 353 features in (+) mode and 558 and 446 features in (-) mode, respectively. A total of 233 metabolites were annotated using the (±) separate polarities and 179 using the (±) switching polarity based on MassBank of North America (MoNA) public MS library and filtered with the Human Metabolome Database (HMDB). Both switching and separate polarity methods performed well regarding response linearities which were investigated by spiking some amino acid compounds into plasma matrix. Conclusions: The polarity switching DIA approach for metabolomics reduced sample consumption and analysis time, but led to fewer detected features and annotations compared to separate polarity runs. These findings support the use of unified nanoLC-MS platforms for integrated multi-omics analysis.