Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The preparation of high-quality mass-spectrometry-compatible samples for comprehensive proteome analysis of ocular samples is critical to elucidate the molecular mechanisms and signaling pathways implicated in health and disease. The described work flow represents a simple yet robust approach to stringent sample preparation steps catered specifically for analysis of mass-limited samples such as ocular micro blood vessels. Although the main aim of the study is to establish an MS-compatible methodology for ocular blood vessels, the described work flow can be broadly applied to various cell-and tissue-based samples.

Generally, individuals new to this method may struggle because the identification and isolation of the chosen short posterior ciliary artery, or SPCA branches, can be challenging. Fresh porcine eyes, together with optic nerve and extraocular tissues, were obtained from the local abattoir immediately post-mortem. Place the eye globe in a dissection chamber containing ice-cold Krebs-Henseleit buffer.

Carefully cut away surrounding muscle and tissues with a pair of sharp Mayo scissors. Make an incision with a scalpel, and cut the globe along the equatorial plane with a pair of sharp Mayo scissors until the eye is separated into the anterior and posterior halves. Remove as much vitreous body as possible from the posterior half of the eye using a pair of standard pattern forceps.

After using dissection pins to carefully pin down the posterior half of the eye, gently cut away the connective tissues surrounding the optic nerve, to expose the underlying retrobulbar vasculature with a pair of student Vannas spring scissors. Isolate the paraoptic and distal short posterior ciliary artery together with the surrounding connective tissues with a pair of type five precision tweezers and Vannas capsulotomy scissors. Gently remove connective tissues from the arterial segments using extra fine tipped tweezers and scissors before rinsing the isolated arteries in ice-cold PBS to remove contaminants and blood residues.

Pool arteries from two eyes to obtain one biological replicate, then weigh the samples using an analytical balance. To each tube, add a mixture of 0.5 and one-millimeter zirconium oxide beads followed by tissue protein extraction reagent. Now, load the sample tubes into a blender homogenizer.

Set the timer to two minutes, set the speed level to six, and start homogenization. After running, check the samples for complete homogenization and keep samples on ice in between each run. Repeat the cycle until samples are completely homogenized.

Carefully pipette homogenate into fresh microcentrifuge tubes. Centrifuge the samples at 10, 000 times G for 20 minutes at four degrees Celsius to pellet insoluble proteins. Carefully pipette the supernatant containing the soluble proteins without touching the pellet layer, and transfer into fresh microcentrifuge tubes.

Add 200 microliters of Extraction Buffer 2A and five microliters of protease inhibitor cocktail to each pellet sample. Then, suspend the pellet several times with a pipette. Use an ultrasonic homogenizer to completely homogenize the pellet.

Set the amplitude to 60 and cycle to one. Use a probe made of titanium, which is appropriate for homogenization of samples with small volumes. Immerse the probe in the pellet and extraction buffer mixture on ice, and press the start button.

Sonicate the sample until the pellet clumps are completely homogenized, pausing for a few seconds between each sonication. Check for complete homogenization visually. Mix the homogenate several times with a pipette to ensure that there are no clumps.

Use centrifugal filter devices with three-kilodalton cutoff for this procedure. Insert the three-kilodalton cutoff filter unit into a microcentrifuge tube. Pipette 200 microliters of sample homogenate to one filter device, and add 200 microliters of deionized water into the same filter.

After capping it securely, place the filter unit into a centrifuge, and spin for 14, 000 times G for 15 minutes at four degrees Celsius. After centrifugation, separate the filter unit containing the sample concentrate from the microcentrifuge tube containing the filtrate, discarding the filtrate. Reconstitute the concentrate by adding 400 microliters of deionized water into the filter unit.

After repeating filtration three times, carefully pipette the cleaned sample concentrate into a clean microtube. Prepare the samples for one-dimensional gel electrophoresis as listed in the text protocol, and mix well with a pipette. Heat the samples at 70 degrees Celsius in a dry block heater for 15 minutes, and cool to room temperature.

Carefully load 50 micrograms of sample per lane using a pipette, as well as the prestained protein standard as a molecular mass marker. Run the gels for approximately 60 minutes at a constant voltage of 175 volts. At the end of the run, carefully remove the gel from the cassette plate using a gel knife, and transfer the gel into a gel staining box.

Perform fixing and staining of the gel as described in the text protocol. Shake the gels in the staining solution overnight for best overall results. Carefully decant the staining solution and replace with 200 milliliters of deionized water before shaking the gels for at least seven to eight hours in water to clear the background.

Excise the protein bands from the gel with clean new microtome blades. Cut the band into small pieces, and carefully transfer the gel pieces into 1.5-milliliter microcentrifuge tubes. Add 500 microliters of destaining solution containing 100-millimolar ammonium bicarbonate and acetonitrile.

Incubate the samples at room temperature for 30 minutes, with occasional shaking. Carefully pipette out the destaining solution. Check visually for any tubes with residual stain, and repeat this step if gel pieces are still stained blue.

Add approximately 400 microliters of freshly prepared DTT solution, and incubate at 56 degrees Celsius for 30 minutes. After discarding the reducing solution with a pipette, add approximately 400 microliters of freshly prepared IAA solution, and incubate in the dark at room temperature for 30 minutes. Remove the alkylating buffer with a pipette and discard.

Add 500 microliters of neat acetonitrile at room temperature for 10 to 15 minutes, until the gel pieces shrink and become opaque. Pipette out the acetonitrile, and air-dry the gel pieces for five to 10 minutes under the hood. Then, pipette 50 microliters of trypsin solution into each tube to completely cover the gel pieces, and incubate the tubes at four degrees Celsius.

After 30 minutes, add sufficient volume of trypsin buffer to completely cover the gel pieces if necessary. Incubate the samples overnight at 37 degrees Celsius. To perform peptide extraction, carefully pipette the extracted peptide solution from the tubes and transfer to clean microtubes.

Dry the supernatant in a centrifugal vacuum evaporator. Add 100 microliters of extraction buffer to each tube with gel pieces, and incubate for 30 minutes with shaking. Pipette the supernatant into the same microtubes containing the extracted peptides according to their respective bands, and dry down in a vacuum centrifuge.

Proceed to peptide purification and liquid chromatography-electrospray ionization MS/MS analyses as described in the text protocol. The total amount of proteins in samples extracted with each type of detergent is depicted here. The highest yield came from tissues extracted with tissue protein extraction buffer, followed by DDM, CHAPS, ASB-14, and the lowest yield from ACN and TFA.

Consistently, the total proteins identified were also the highest in the tissue protein extraction buffer extracted sample, and followed same trend as the total yield. Shown here is the comparison of the one-dimension gel electrophoresis protein profiles of SPCA, before and after being subjected to the optimized sample preparation and cleaning steps. Overall, a high degree of smearing and poor separation of the protein bands was observed at lane three.

This profile demonstrates that the samples may contain extraction reagent, and also contaminants such as lipids and cellular debris. However, the SPCA samples that were separated into supernatant and pellet, and then subjected to the optimized protocol, resulted in exemplary one-dimensional gel electrophoresis profiles. The optimized method for rapid, robust, and efficient protein extraction from ocular microvessels can also be readily applied to other tissue-based samples.

Shown here are protein profiles of the supernatant and the pellet of murine brain and cardiac tissue samples. While attempting this procedure, it is important to remember to subject the samples to complete homogenization, to separate the supernatant from the pellet, and to remove the contaminants and the extraction reagents. After its development, this technique paved the way for researchers in the field of experimental and translational ophthalmology to thoroughly explore the proteome of oscular vasculature using porcine eyes as a model.