Microwave & magnetic proteomics of macrophages from patients with HIV-associated cognitive impairment

<div><p>Objective</p><p>HIV-infected monocytes can infiltrate the blood brain barrier as differentiated macrophages to the central nervous system, becoming the primary source of viral and cellular neurotoxins. The final outcome is HIV-associated cognitive impairment (HACI), which remain prevalent today, possibly due to the longer life-span of the patients treated with combined anti-retroviral therapy. Our main goal was to characterize the proteome of monocyte-derived macrophages (MDM) from HACI patients, and its association with their cognitive status, to find novel targets for therapy.</p><p>Methods</p><p>MDM were isolated from the peripheral blood of 14 HIV-seropositive women characterized for neurocognitive function, including: four normal cognition (NC), five asymptomatic (A), and five with cognitive impaired (CI). Proteins from macrophage lysates were isobaric-labeled with the microwave and magnetic (M2) sample preparation method followed by liquid chromatography-tandem mass spectrometry-based protein identification and quantification. Differences in protein abundance across groups classified by HACI status were determined using analysis of variance.</p><p>Results</p><p>A total of 2,519 proteins were identified with 2 or more peptides and 28 proteins were quantified as differentially expressed. Statistical analysis revealed increased abundance of 17 proteins in patients with HACI (p<0.05), including several enzymes associated to the glucose metabolism. Western blot confirmed increased expression of 6-Phosphogluconate dehydrogenase and L-Plastin in A and CI patients over NC and HIV seronegatives.</p><p>Conclusions</p><p>This is the first quantitative proteomics study exploring the changes in protein abundance of macrophages isolated from patients with HACI. Further studies are warranted to determine if these proteins may be target candidates for therapy development against HACI.</p></div

Validation of proteins identified by western blot.

<p>(A) Proteins identified by TMT labeling were tested by western blot from MDM lysates of the same patients whose samples were used for proteomics. (B) Densitometry analyses for the western blots were normalized against GAPDH. The statistic analysis between the three groups of patients was performed using One-way ANOVA with a significance of *p<0.05. For Plastin-L, there were significant differences between NC vs CI (p = 0.0316), and between ANI vs CI (p = 0.042).</p

Predicted network of interactions between the proteins identified in macrophages from HACI patients.

<p>(A) Blue proteins are from our dataset and the grey colored proteins are the ones that connect the proteins in our dataset according to IPA software. (B) The lower panel shows the pattern of increase/decrease followed by vimentin, EEF1 and cathepsin B among the groups of HACI patients.</p

Differentially expressed proteins by TMT and mass spectrometry.

<p>A-D are proteins involved in the glycolysis pathway. E and F are the proteins involved with cell protection. G is the protein involved with neurotoxicity. H, and P are the proteins involved in oxidative stress and protein synthesis. From I to O are the proteins involved with the cell structure and motility. *p<0.05; **p<0.01.</p

List of proteins identified by TMT labeling and differentially expressed among the HACI groups.

<p>List of proteins identified by TMT labeling and differentially expressed among the HACI groups.</p

Patient samples used for TMT labeling.

<p>Patient samples used for TMT labeling.</p