SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Summary

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

Abstract

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Introduction

Many human diseases, including viral infections, are often associated with distinctive cellular processes that take place in and around the affected cells. Proteins, often acting as the ultimate cellular effectors, mediate these processes. Analysis of the proteins often can provide invaluable information as to the local environment of affected cells and help us to understand the underlying mechanism of disease pathogenesis. Among various protein analysis techniques, proteomics holds particularly great promise. As a powerful, large-scale tool, proteomics can provide a systemic understanding of cellular processes, particularly in the area of the function and interaction of proteins. Analyzing specific proteins is made simpler through the development of labeling techniques, which allow investigators to monitor the expression of cellular components, particularly proteins, in the site of investigation. Although many proteomic analyses have been performed at cellular proteome scale, proteomic characterizations on subcellular compartments have proved to be particularly informative¹. This is exemplified well in the studies of HIV-1 infection.

Exosomes, 30-100 nm membrane vesicles secreted by a wide range of cell types^2,3, are critical components of intercellular communication and molecular transport. They were previously discovered to play important roles in the HIV-1 budding process^4,5. By combining proteomic analysis with functional dissection, we found that exosomes released from HIV-1 infected cells are composed of a unique and quantitatively different protein signature and harbor regulatory molecules that impact cellular properties on neighboring receptive cells, including cellular apoptosis and proliferation⁶. The methods are described in this protocol, namely SILAC (stable isotope labeling by amino acids in cell culture)⁷ based proteomic characterization of exosomes from HIV-1 infected cells. Similar approaches can be applied to better understand other subcellular compartments during pathogenesis by adjusting the experimental stress to the specific compartment or fraction of interest and making necessary changes to the described procedures.

Given the recent development of quantitative proteomic methods, there are many to choose from when selecting the most efficient method for a particular experiment. Among these are the chemical-based iTRAQ (isobaric tags for relative and absolute quantification)⁸ and the label-free MRM (multiple reaction monitoring)⁹ techniques. Both methods are powerful tools and are good choices for specific settings. For a typical laboratory mainly working with cell lines, however, these two methods have relatively higher costs and are more time-consuming when compared to the SILAC based method. SILAC is a metabolic based labeling technique that incorporates nonradioactive isotopic forms of amino acids from the culture media into cellular proteins. Typically, SILAC experiments start with two cell populations, for example, infected and uninfected. Each is differentially labeled in its specific isotopic environment until full labeling is achieved. The labeled exosomes of these cells are then subjected to protein extraction. Once extracted, the labeled exosomal proteins are analyzed using liquid chromatography tandem mass spectroscopy¹⁰. Finally, the mass spectrometry results and significantly labeled proteins are subjected to statistical and bioinformatics analyses as well as rigorous biochemical verification. Our previous investigative reports suggest that the SILAC/exosome procedures are more appropriate for cell lines than primary cells, as cell lines are usually in an active proliferating state for efficient isotopic labeling,

Protocol

1. Cell Culture and HIV-1 Infection

NOTE: Before starting experiments, it is recommended to check the cells' viability through Trypan Blue staining¹¹ and their proliferation through a MTT assay¹². It is also critical to use newly prepared SILAC medium. Various cell lines can be used, as long as they are in an actively proliferative stage, and are susceptible to HIV-1 infection, or the test condition of choice. In this protocol, use H9 cell line as the example.

1. Seed 2 x 10⁶ H9 cells into each cell culture flask. Grow one group in 10 ml labeled RPMI 1640 medium containing 10% dialyzed fetal bovine serum (FBS), 100 mg/L ¹³C₆ L-lysine and 100 mg/L ¹³C₆¹⁵N₄ L-arginine. Grow the other group in 10 ml unlabeled RPMI 1640 medium, with 10% dialyzed FBS, 100 mg/L L-Lysine and 100 mg/L L-Arginine.
2. Grow the cells for six doublings at which point the proteins of the cells in the labeled medium are practically completely labeled (>99%) with heavy amino acids. Add fresh media or change media regularly (every 1-3 days depending on the type of cell. For H9 cell, change medium every 3 days). At the end of labeling, increase culture volume (e.g. ~30 ml) to accommodate the growth of more cells.
   NOTE: Determine exact cell doubling time at the beginning of the experiment via the Trypan Blue staining and cell counting.
3. Infect the labeled cells with HIV-1_NL4-3 using standard HIV-1 infection protocol¹³ for 48 hr. Incubate the cells with appropriate amounts of virus with a multiplicity of infection (MOI) around 0.3. Check HIV-1 infection by p24 quantification of the culture supernatants using an HIV-1 p24 antigen ELISA kit. Perform an immunofluorescence assay¹⁴ to confirm successful infection of cells. Keep growing the unlabeled cells in unlabeled medium without HIV-1 infection.
4. Harvest the supernatants of both groups at the end of infection (step 2.1).

2. Exosome Isolation

NOTE: Through a series of ultracentrifugation steps, exosomal fractions from culture supernatants are enriched¹⁵. Perform all the following steps at 4 °C, with an ultracentrifuge rotor that can reach a speed of at least 100,000 x g.

1. Collect the supernatants of the cultures in 50 ml conical tubes, avoiding cells. Centrifuge them for 10 min at 300 x g to remove remaining cells.
2. Collect the supernatants in new 50 ml conical tubes and centrifuge them for 10 min at 2,000 x g to remove dead cells. Transfer resulting supernatants to commercial rotor-compatible tubes that are able to withstand ultracentrifugation.
3. Be sure to balance the ultracentrifuge tubes. Centrifuge the tubes for 30 min at 10,000 x g to remove cell debris.
4. Collect the supernatants in ultracentrifuge tubes and centrifuge for 70 min at 100,000 x g. Discard the supernatants.
5. Resuspend the exosome-rich pellets in 5 ml of fresh PBS. Transfer the solutions to fresh ultracentrifuge tubes and centrifuge again for 70 min at 100,000 x g.
6. Discard supernatants. To store the exosomes long-term, resuspend the pellets in 50 µl of PBS and store at -80 °C. Alternatively, proceed directly to protein extraction as shown below.

3. Protein Extraction and Preparation

1. Dissolve isolated exosomal pellets in 100-200 µl RIPA lysis and extraction buffer with added protease inhibitor cocktails. The buffer is composed of 25 mM Tris-hydrochloride, 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS (sodium dodecyl sulfate), at pH 7.6. The protease inhibitor cocktails should contain a variety of protease inhibitors, such as aprotinin, bestatin, leupeptin, pepstatin A and EDTA (ethylenediaminetetraacetic acid), that can inhibit a full range of proteases.
2. Centrifuge the dissolved solutions for 10 min at 13,000 x g (4 °C), and transfer the cleared supernatants to new 1.5 ml microcentrifuge tubes.
3. Quantify protein concentration of each sample of exosomes using bicinchoninic acid (BCA) or Bradford assay¹⁶.
4. Mix an equal amount of proteins (2 µg) from labeled and unlabeled samples and run the equal mixture on a 4-20% SDS-PAGE gel at 120 mA/200 V for 30 min.
5. Stain gel with Coomassie Blue followed by destaining¹⁷.
6. Using a razor blade, cut the sample lane from the gel. Then cut the gel lane into 10-15 equal pieces. Put each piece into a fresh 1.5 ml microcentrifuge tube for a total of 10-15 tubes.
7. Immerse cubes in 25 mM NH₄HCO₃ in 50% acetonitrile, vortex, and discard supernatants. Repeat twice. Dry cubes in a vacuum concentrator^18,19.
8. Rehydrate cubes with 10 mM dithiothreitol (DTT), vortex, and centrifuge briefly. Incubate at 56 °C for 1 hr. Discard supernatant.
9. Immerse gel cubes in 55 mM iodoacetamide, vortex, and spin. Incubate at room temperature in the dark for 45 min. Discard supernatant.
10. Immerse cubes in 25 mM NH₄HCO₃, vortex, spin, and discard supernatant.
11. Immerse cubes in 25 mM NH₄HCO₃ in 50% acetonitrile, vortex, and spin. Repeat 3.9 and 3.10.
12. Dry the cubes in a vacuum concentrator. Add 25 µl sequencing grade modified trypsin in 25 mM NH₄HCO₃, incubate at 4 °C for 30 min, and discard the excess solution. Immerse cubes in 25 mM NH₄HCO₃ without trypsin, and incubate overnight at 37 °C.
13. Briefly spin cubes down and transfer the resulting peptide extract to a new tube. Add 30 µl of 5% formic acid in 50% acetonitrile to the cubes, vortex for 30 min, and spin. Combine the supernatant with the extract. Dry the peptide extracts with a vacuum concentrator to less than 5 µl.
14. Submit samples to mass spectrometry core facility for LC-MS/MS analysis. The MS analysis data, which can be generated from open source software, typically includes an accession number for each protein identified, labeled/unlabeled ratios and the number of unique peptides identified.

4. Western Blotting Verification

NOTE: Western blotting is recommended to verify mass spectrometry results.

1. Follow standard western blotting protocols and ensure that equal amounts of protein from each group are loaded. Use antibodies against proteins of interest (e.g. annexin A5, lactate dehydrogenase B chain) identified by MS. Western blotting detection, along with densitometry analysis, can reaffirm that MS protein identification and quantification are correct.

5. Proteomic Data Analysis

NOTE: The data quality assessment, data pretreatment, calculation and determination of significant protein candidates are done separately for each MS replicate. Once above analyses are completed, the analyzed data from replicates are compared and combined^6,20,21.

1. Assess the quality of MS data.
   1. First, log2 transform the SILAC-MS labeled/unlabeled ratios of quantified proteins in a spreadsheet program.
   2. In scientific graphing and statistical software, group the ratios into 40-100 ratio bins and plot the number of ratios per bin to generate a histogram. A normal distribution of histogram indicates good quality of MS data⁶. Do this step for all the MS replicates.
2. To increase the confidence and accuracy of the MS peptide ratios, consider removing proteins that have less than two quantified peptides.
3. To determine significantly up- and down-regulated protein candidates, use the following steps to calculate significance thresholds²².
   1. In scientific graphing and statistical software, generate a non-linear regression or curve-fit to the frequency distribution data. This step yields values that can be used to calculate the cutoff values. Calculate the cut-off values as median ± 1.96 σ for 95% confidence limits or 2.56 σ for 99% confidence limits.
      NOTE: Specific details of calculating the cutoffs can be found at Emmott et al.²².
   2. Select the protein candidates whose ratios are either greater than 1.96 (or 2.56) standard deviations above the median (significantly over-expressed) or lower than 1.96 (or 2.56) standard deviations below the median (significantly under-expressed).
4. Compare the replicates of the above-identified significant candidates to achieve consistency. Ensure that they meet the following criteria to pass this final selection step: candidates must be consistently identified in all replicates; and replicates of a candidate must be consistent in their direction of regulation (preferably, at least 2 out of 3 replicates of a candidate should either be up-regulated or down-regulated).
5. Finalize data for candidates that meet the above-mentioned criteria by merging their replicates' data.

6. Bioinformatics Verification and Characterization

NOTE: Existing genomic and bioinformatic information offers a wealth of information for almost every protein. Data mining and bioinformatics analysis on that information can help in gaining a great deal of insight into the property and functions of the significant candidates. This process is usually necessary to design proper downstream wet-lab experiments.

1. Using their GenBank Accession numbers or UniProt IDs, search the candidates against current exosome databases (Exocarta²³, Evpedia²⁴) to verify that the candidate proteins have been previously found in exosome. This step adds a layer of confidence that the candidates are indeed in exosomes.
   1. Access http://www.exocarta.org/, click “Query” and input either the gene or protein name/accession number. A summary page will appear and the evidence in exosome will be shown, provided if there is any.
2. Search the candidates against the HIV-1 and the Human Protein Interaction Database²⁵. Alternatively, search the specific databases that can provide information about the interaction of the candidate proteins and the test condition.
   1. Access the database at www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions. On the ‘protein domain name’ entry, enter the candidates’ 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.
3. Import GenBank Accession numbers or UniProt IDs of the candidates into GO analysis software (such as FunRich, Functional Enrichment analysis tool²⁶) and run the software.
   1. Download the software at http://www.funrich.org/. After installation, open the software, under enrichment analysis, click “Add Data Set”, and upload the list of protein/genes. Next, select the chart type to visualize the GO analysis.
      NOTE: GO analysis results will be visualized in the form of pie charts. This step helps us to gain global insight associated with the candidates in the areas of Biological Process (BP), Cellular Component (CC), and Molecular Function (MF).
4. Access DAVID at http://david.ncifcrf.gov/²⁷, click the “Functional Annotation Tool” on its website. Enter the candidate list, select the “Identifier” of the gene such as the “UniProt ID”, select “Gene List” and search. The enriched GO terms, p-values, and other parameters can be found by clicking the “Annotation Summary Results” page under the “Gene_Ontology” category.
5. To investigate potential interactions of the candidates with other proteins, use openly accessible STRING database²⁸ to elucidate potential protein-protein interactions and possible biochemical pathways.
   NOTE: GO Analysis and Functional Annotation (Section 6.3-6.4) can also be performed using the latest STRING software.
   1. Access the database at http://string-db.org/. 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 (physical) and indirect (functional) associations. The top ten known matches for the exosomal candidate will be displayed and should be considered for significant candidate selection.
      NOTE: A great deal of information associated with the candidates can be analyzed using the steps above. The most significant candidates may be chosen for further downstream molecular and biochemical analysis.

Results

Figure 1A is a flowchart outlining the SILAC labeling procedure²¹. In order to purify the exosomes, the samples must be spun down via centrifuge. Figure 1B shows the steps of exosome purification by serial ultracentrifugation²¹. Once purified, the exosomes are subject to experimental proteomic analysis as outlined in the procedure. 

Figure ...

Discussion

In the procedures described in this paper, we demonstrated the application of the SILAC technique to investigate the effect of HIV-1 infection on the host exosomal proteome. Initially, uninfected and HIV-1 infected cells are differentially isotope-labeled. The differentially labeled exosomes are then purified before performing protein extraction. Next, liquid chromatography-tandem mass spectrometry is employed to analyze the exosomal proteome. Finally, the resulting mass spectrometry data and potential candidate proteins...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
H9 cell line	ATCC	HTB-176
Trypan Blue	Thermo Fisher	15250061
MTT assay kit	Thermo Fisher	V13154
Dialyzed fetal bovine serum (FBS)	Thermo Fisher	26400044
SILAC Protein Quantitation Kit – RPMI 1640	Thermo Fisher	89982	DMEM version (89983)
L-Arginine-HCl, 13C6, 15N4 for SILAC	Thermo Fisher	88434
L-Lysine-2HCl, 13C6 for SILAC	Thermo Fisher	88431
HIV-1 NL4-3	NIH AIDS Reagent Program	2480
Alliance HIV-1 p24 Antigen ELISA kit	PerkinElmer	NEK050001KT
Refrigerated super-speed centrifuge	Eppendorf	22628045
Refrigerated ultracentrifuge	Beckman Coulter	363118	Should be able to reach 100,000 x g
50 mL Conical Centrifuge Tubes	Thermo Fisher	14-432-22
Ultracentrifuge Tubes	Beckman Coulter	326823
SW 32 Ti Rotor	Beckman Coulter	369694
RIPA buffer	Thermo Fisher	89900
Protease Inhibitor Cocktails	Thermo Fisher	78430
ThermoMixer	Eppendorf	5384000020
BCA Protein Assay Kit	Thermo Fisher	23250
Spectrophotometer	Biorad	1702525
SDS PAGE Gel apparatus	Thermo Fisher	EI0001
Novex 4-20% Tris-Glycine Mini Gels	Novex	XV04200PK20
Gel staining reagent	Sigma Aldrich	G1041
Sequencing Grade Modified Trypsin	Promega	V5111
SpeedVac Concentrator	Thermo Fisher	SPD131DDA
Antibody to human annexin A5	Abcam	ab14196
Antibody to human lactate dehydrogenase B chain	Abcam	ab53292
Graphing and Statistical Software	Systat	SigmaPlot	Or GraphPad Prism
Quantitative proteomics software suite	Max Planck Institue of Biochemistry	Maxquant
Software and databases	Various vendors		Refer to main text for details