Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria

Graphical abstract


Method details
Isolation of pure mycobacterial and Gram-positive bacteria (Bacillus subtillis) membrane vesicles involves growing the bacteria in liquid media to mid-logarithmic phase, followed by clarification of the cell supernatant by sequential filtration and concentration. The concentrate is then ultracentrifuged and the pellet is submitted to density gradient ultracentrifugation to obtain the pure membrane vesicle fractions [1][2][3]. Proteins in the purified fractions are then acetone precipitated and in-solution digested with trypsin. Generated peptides are separated by nano-LC and analyzed by mass spectrometry for protein identification [4,5].
Step 1: isolation of membrane vesicles

Materials
Minimal media (MM): Minimal media or chemically defined media is used to reduce the number of components in the culture supernatant. To approximately 3.9 L of double distilled water in a 4 L beaker, add 4 g KH 2 PO 4 , 10g Na 2 HPO 4 and 2 g asparagine. Adjust the pH to 7.0 using HCl. Filtersterilize (0.22 mm) the solution. Add 56 ml of filter-sterilized 50% (v/v) glycerol, and 0.4 ml of filtersterilized 500 mg/l ferric ammonium citrate, and 0.4 ml of 5 mg/l CaCl 2 , and 0.4 ml of filter-sterilized 1 mg/l ZnSO 4  A B S T R A C T Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicleassociated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are: Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant. The use of an ultrafiltration system, which allows concentrating larger volumes.
In solution digestion of proteins followed by peptides purification by ziptip.  Gram-positive bacteria incubate the flask at 37 8C overnight ($18 h) in an orbital shaker at 200 rpm. 5. Filter the cultures through a 0.45-mm-pore size filter. 6. Filter the clarified supernatants through a 0.22-mm-pore size filter. 7. Concentrate the supernatant using an Amicon Ultrafiltration system with a 100-kDa-exclusion filter. Leave 10-15 ml of liquid. 8. Rinse the filter with the remaining supernatant and recover the concentrate. 9. Sequentially centrifuge the concentrate at 4000 and 15,000 Â g (15 min, 4 8C) to remove aggregates. 10. Ultracentrifuge the remaining supernatant at 100,000 Â g for 1 h at 4 8C to obtain the membrane vesicle pellet.
Step 2: purification of membrane vesicles by density gradient ultracentrifugation ranging from 10 to 35% (w/v). 4. Ultracentrifuge the gradient at 140,000 Â g for 16 h at 4 8C using a swinging rotor. 5. Take 1 ml fractions from the top of the gradient to the bottom and keep the 3 and 4 fractions, which contain the pure membrane vesicles. If it is the first time isolating membrane vesicle from a bacteria it is recommended to analyze each fraction for the presence of membrane vesicles. We recommend submitting samples to electron microcopy analysis, performing an analysis by immunoblot with specific antibodies against vesicle-associated proteins, or metabolic labeling of vesicles to identify the radio-labeled vesicle fractions. 6. Pool the selected vesicle-containing fractions. 7. Dilute the pooled fractions in DPBS and centrifuge at 38,400 Â g for 2 h to remove the Optiprep. 8. Suspend the vesicle pellet in 0.5 ml of DPBS.

Step 3: Protein digestion
As with any sample preparation method for MS analysis, special attention must be paid to avoid loss or contamination of the samples during processing. Please be very careful not to use bare hands, loose hair, dirty glass-and plastic-ware, and always wear gloves (powder free and rinsed with water and ethanol before use) to minimize contamination by keratins, or these proteins will overwhelm low level protein samples and preclude successful analysis of the proteins of interest.

Materials
Mass Spec-grade acetone at À20 8C. 25 mM ammonium bicarbonate: dissolve 0.2 g of ammonium bicarbonate in 100 ml of Mass Specgrade water. 10 mM DTT: dissolve 15 mg of dithiothreitol (DTT) in 10 ml of 25 mM ammonium bicarbonate. 10 mM iodoacetamide: dissolve 18 mg of DTT in 10 ml of 25 mM ammonium bicarbonate. 50 ng/ml trypsin solution: dissolve 100 mg of trypsin gold, mass spectrometry grade (Promega), in 2 ml of 25 mM ammonium bicarbonate. Microcentrifuge with speed up to 13,000 rpm. Thermomixer with 1.5 ml tubes block (Eppendorf). Centrifugal vacuum concentrator with a rotor to accommodate 1.5 ml tubes.
Note: All solutions and buffers should be prepared in Mass Spec-grade water. Reconstituted trypsin can be stored at À20 8C for up to 1 month. For long-term storage, freeze reconstituted trypsin at À70 8C. Before use, thaw the reconstituted trypsin at room temperature, placing on ice immediately after thawing. To maintain sufficient enzymatic activity, limit the number of freeze-thaw cycles to 5. Iodoacetamide binds covalently with the thiol group of cysteines, thus avoiding formation of disulfide bonds. 7. Add 40 ml of trypsin solution and incubate at 37 8C overnight ($16 h) with gentle agitation using a thermomixer at 300 rpm. Cap the tubes tightly and cover with parafilm to avoid evaporation. 8. After completing the digestion, dry the samples using a centrifugal vacuum concentrator.
Step 4: peptide analysis and protein identification The following instructions assume the use of a NanoLC Plus (Eksigent) HPLC system coupled directly to an LTQ XL linear ion trap mass spectrometer (ThermoFinnigan). Similar instruments can also be used.
Buffer A: 2% Mass Spec-grade acetonitrile, 0.1% formic acid in Mass Spec-grade water. Buffer B: 0.1% formic acid in Mass Spec-grade acetonitrile. NanoLC system coupled to a biological mass spectrometer able to perform MS/MS analysis. Database engine such as Mascot (Matrix Science) for protein identification. 4. Load the peptides onto a 0.3 mm Â 5 mm C18 precolumn (New Objective) at a flow-rate of 3 ml/min. 5. Reverse the flow through the precolumn and elute the peptides with a gradient starting at 95% buffer A and ending at 90% buffer B. The gradient is delivered over 120 min at a flow-rate of 200 nl/ min through a 75 mm Â 15 cm fused silica capillary C18 HPLC column (LC Packings) to a stainless steel Nanobore emitter (Proxeon). The electrospray needle can vary depending on the ion source of the mass spectrometer. Fig. 1 shows a typical chromatogram obtained with the described system. 6. Use a database search engine such as Mascot (Matrix Science) for database searching and protein identification.