The overall goal of this procedure is to characterize the exosomal proteome from HIV-1 infected cells. This method can help answer the key question in the exosome biology field such as the effect of external stresses on the composition of the exosome proteome. The main advantage of this technique is that it can be learned even with a limited background in proteomics and exosome biology.

Though this method can provide insight into HIV infection, it can also be applied to other disease studies such as bacterial infections. The H9 cell line is used in this experiment although various cell lines can be used as long as they are in actively proliferating stage and are susceptible to the test condition of choice. Seed two times 10 to the sixth stage nine cells into each of two cell culture flasks.

Grow one population in 10 milliliters of labeled medium containing 10%stylized FBS, 100 milligrams per liter C13-labeled L-lysine, and 100 milligrams per liter C13 and N15-labeled L-arginine. Grow the other population in 10 milliliters of unlabeled medium with 10%stylized FBS, L-lysine, and L-arginine. Grow the cells for six doublings at which point the proteins of the cells in the labeled medium are more than 99%labeled with heavy amino acids.

Add fresh media or change media at regular time intervals depending on the type of cells. At the end of labeling, increase the culture volume to 30 milliliters to accommodate the growth of more cells. Infect the labeled cells with the NL4-3 strain of HIV-1 using a standard HIV-1 infection protocol.

Add an appropriate amount of virus to the cells and incubate for 48 hours while the unlabeled cells continue to grow in unlabeled medium without HIV-1 infection. At the end of HIV-1 infection, the supernatants from both cell populations are harvested for exosome isolation. Collect the supernatants of the cultures in 50-milliliter conical tubes avoiding the cells.

Centrifuge for 10 minutes at 300 times g and four degrees Celsius to remove remaining cells. Collect the supernatants in new 50-milliliter conical tubes and centrifuge for 10 minutes at 2, 000 times g to remove dead cells. Transfer the resulting supernatants to commercial rotor-compatible tubes that are able to withstand ultracentrifugation.

Be sure to balance the ultracentrifuge tubes. Centrifuge the tubes for 30 minutes at 10, 000 times g to remove cell debris. Collect the supernatants in new ultracentrifuge tubes.

Centrifuge for 70 minutes at 100, 000 times g. Discard the supernatant. Resuspend each exosome-rich pellet in five milliliters of fresh PBS and transfer the solution to a fresh ultracentrifuge tube.

Centrifuge again for 70 minutes at 100, 000 times g. After the final spin, discard the supernatant. The cell pellets are now ready for protein extraction.

To begin this procedure, dissolve each isolated exosomal pellet in 100 to 200 microliters of fer-pa-lai-ses and extraction buffer with added protease inhibitors that can inhibit a full range of proteases. Centrifuge the dissolved solutions for 10 minutes at 13, 000 times g and four degrees Celsius. Transfer the cleared supernatants to new 1.5-milliliter microcentrifuge tubes.

After quantifying the protein concentration of each sample by standard assays, mix equal amounts of proteins from the labeled and unlabeled samples. Run the equal mixture on a four to 20%SDS-PAGE gel for 30 minutes. Stain the gel with Coomassie blue followed by destaining.

Using a razor blade, cut the sample lane from the gel, then cut the gel lane into 10 to 15 equal pieces. Put each piece into a fresh 1.5-milliliter microcentrifuge tube for a total of 10 to 15 tubes, and continue with the protein extraction procedure as described in the text protocol. The procedures for proteomic data analysis will not be demonstrated but are summarized in this flowchart.

The extracted proteins are first analyzed by liquid chromatography, tandem mass spectroscopy, and the quality of the data is assessed. Next, the data is pretreated by removing proteins that have less than two quantified peptides. Significantly upregulated and downregulated proteins are then identified.

Finally, the replicates of the significant candidates are compared to achieve consistency, and the data from all replicates are merged. To begin the characterization of the identified proteins, use their GenBank accession numbers or UniProt IDs to search against current exosome databases. ExoCarta will be used in this demonstration.

Click query and input either the gene or protein name or accession number. Any evidence in exosomes verifies that the candidate proteins have been previously found in exosomes and it adds a layer of confidence that the candidates are indeed in exosomes. Next, search the candidates against the HIV-1 and human protein interaction database.

On the protein domain name entry, enter the candidate's names or accession numbers, and click search. The search results can provide insight into the interactions between HIV-1 and the protein candidates, and suggest which of the candidates might be truly HIV-1-associated. The third step is to gain global insight on the candidates using a gene ontology or GO analysis software such as fun-brich.

Under enrichment analysis, click add dataset and upload the GenBank accession numbers or UniProt IDs of the candidates, then select the chart type to visualize the GO analysis. GO analysis result are visualized in the form of pie charts that provide information about the candidate proteins in the areas of biological process, cellular component, and molecular function. The next step is to identify statistically overrepresented GO terms via DAVID analysis.

Click the functional annotation tool, enter the candidate list, and select the identifier of the gene such as the UniProt ID.Select gene list and search. When the search is complete, click the annotation summary results page under the gene ontology category to view the enriched GO terms, p-values, and other parameters. Finally, investigate potential protein-protein interactions and possible biochemical pathways using the openly accessible STRING database.

Input the protein ID or sequence into the designated search box and select the correct species for analysis. Click search. The results will give information on both direct and indirect associations.

The top 10 known matches for the exosomal candidate displayed in the results should be considered for a significant candidate selection. Representative results from a DAVID analysis of a set of 14 proteins are shown. The biological process enrichment indicated that cell death-related processes are significantly enriched.

The cellular component enrichment shows many proteins with an intracellular origin, and the molecular function enrichment identified a role in protein binding for most of the candidates. L-lactate dehydrogenase B chain or LDHB was selected as the most significant candidate, and STRING analysis identified the top 10 interacting partners of LDHB. The majority of LDHB partners were also functionally and locationally related to exosomes in HIV-1.

Once mastered, this technique can be done in one week if it is performed properly. While attempting this procedure, it is important to remember to perform each step at the right temperature and condition. Following this procedure, other methods such as affinity isolation can be performed in order to confirm the predictions of bioinformatic analysis.

After its development, this technique paved the way for researches in the exosomal field to explore the changes in the exosomal proteome associated with HIV-1 infections in biology. After watching this video, you should have a good understanding of how to isolate exosome from cell culture, analyze their proteomes, and investigate their association with HIV. Don't forget, work with HIV or other infectious agent can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure.