SWATH-MS and MRM: Quantification of Ras-related proteins in HIV-1 infected and methamphetamine-exposed human
Katarzyna Macur1,2, Sarah Zieschang1, Shulei Lei1
1Department of Pharmacology and Experimental Neuroscience, School of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Abstract:
HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be enhanced by exposure to methamphetamine (Meth). In this study, we sought to identify the underlying mechanisms of this effect by quantifying the effect of Meth on the proteome of HIV-1-infected macrophages using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) approach. The analyses identified several members of the Rab family of proteins as being dysregulated by Meth treatment, which was confirmed by bioinformatic analyses that indicated substantial alteration of vesicular transport pathways. Validation of the SWATH-MS was performed using an MRM based approach, which confirmed that Meth exposure affects expression of the Rab proteins. However, the pattern of expression changes were highly dynamic, and displayed high donor-to-donor variability. Surprisingly a similar phenomenon was observed for Actin. Our results demonstrate that Meth affects vesicular transport pathways, suggesting a possible molecular mechanism underlying its effect on HIV infection hMDM and a potential broader effect of Meth on cellular homeostasis.
Insights
Methamphetamine (Meth) alters Rab protein expression in HIV-1-infected macrophages, impacting vesicular transport. This study reveals potential mechanisms for Meth’s enhancement of HIV infection and disruption of cellular balance.
Area of Science:
- Proteomics
- Virology
- Cellular Biology
Background:
- Human immunodeficiency virus type 1 (HIV-1) infection of macrophages is complex.
- Methamphetamine (Meth) exposure is known to enhance HIV-1 infection.
- The precise molecular mechanisms underlying Meth's effect on HIV-1-infected macrophages remain incompletely understood.
Purpose of the Study:
- To investigate the proteomic changes in HIV-1-infected macrophages upon Meth exposure.
- To identify specific protein families and cellular pathways affected by Meth.
- To elucidate the molecular mechanisms by which Meth influences HIV-1 infection and macrophage function.
Main Methods:
- Employed sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to quantify proteomic alterations.
- Utilized bioinformatic analyses to interpret proteomic data and identify affected pathways.
- Validated key findings using a multiple reaction monitoring (MRM) based approach.
Main Results:
- Meth exposure significantly dysregulated Rab family proteins in HIV-1-infected macrophages.
- Bioinformatic analysis indicated substantial alterations in vesicular transport pathways.
- Expression changes in Rab proteins and Actin were dynamic and showed high inter-donor variability.
Conclusions:
- Methamphetamine significantly impacts vesicular transport pathways in HIV-1-infected macrophages.
- These findings suggest a molecular mechanism for Meth's enhancement of HIV infection.
- Meth may broadly affect cellular homeostasis in addition to its specific effects on HIV infection.
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