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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The proteome signature of the inflammatory breast cancer plasma membrane identifies novel molecular markers of disease.

Inflammatory Breast Cancer (IBC) is the most lethal form of breast cancer with a 35% 5-year survival rate. The accurate and early diagnosis of IBC and the development of targeted therapy against this deadly disease remain a great medical challenge. Plasma membrane proteins (PMPs) such as E-cadherin and EGFR, play an important role in the progression of IBC. Because the critical role of PMPs in the oncogenic processes they are the perfect candidates as molecular markers and targets for cancer therapies. In the present study, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) followed by mass spectrometry analysis was used to compare the relative expression levels of membrane proteins (MP) between non-cancerous mammary epithelial and IBC cells, MCF-10A and SUM-149, respectively. Six of the identified PMPs were validated by immunoblotting using the membrane fractions of non-IBC and IBC cell lines, compared with MCF-10A cells. Immunohistochemical analysis using IBC, invasive ductal carcinoma or normal mammary tissue samples was carried out to complete the validation method in nine of the PMPs. We identified and quantified 278 MPs, 76% of which classified as PMPs with 1.3-fold or higher change. We identified for the first time the overexpression of the novel plasminogen receptor, PLGRKT in IBC and of the carrier protein, SCAMP3. Furthermore, we describe the positive relationship between L1CAM expression and metastasis in IBC patients and the role of SCAMP3 as a tumor-related protein. Overall, the membrane proteomic signature of IBC reflects a global change in cellular organization and suggests additional strategies for cancer progression. Together, this study provides insight into the specialized IBC plasma membrane proteome with the potential to identify a number of novel therapeutic targets for IBC.