1 Tissue Preparation
a) Mince the tissue with scissors sterilized with 70% ethanol for at least 1 min. Flush with argon gas before closing the cap of tube to prevent lipid oxidation.
b) Wrap tubes containing weighed tissue samples in parafilm, so the tube cap cannot open during the preparation phase.
c) Using long tweezers, place tubes in liquid nitrogen for 10 min.
d) Transfer tubes to heated water bath of 40 °C for 10 min.
e) Repeat four more times. The process of a hot bath and liquid nitrogen will flash freeze and melt the samples, thus facilitating breaking the phospholipid bilayer cell membrane and allowing for lipids to be more easily solubilized in the organic phase.
2 Lipid Extraction
a) Add 5 mL of LC–MS grade chloroform and 5 mL of LC–MS grade methanol into an amber 12 mL glass vial for a 1:1 (v/v) solution.
b) Measure 220.36 mg of BHT in 10 mL of LC–MS grade water. Take 1 μL of that solution and add it to the previous solution of 10 mL of LC–MS grade methanol:chloroform (1:1) for a final concentration of 10 μM BHT in methanol:chloroform (1:1).
c) Keep the tubes containing lipid samples in ice buckets.
d) Flush lipid samples with argon gas frequently to prevent lipid oxidation.
e) Sterilize handheld homogenizer tip with 70% ethanol.
f) In order to ensure extraction efficiency, an external standard, e.g., 10 pmol of PC (12:0/13:0) can be premixed with sample prior to lipid extraction.
g) Add 500 μL of the methanol:chloroform (1:1) with 10 μM BHT into the tubes containing samples. Homogenize for 2 min. Flush with argon gas afterward. Keep the sample in an ice bucket for as much as possible.
h) Add 300 μL of pure LC–MS grade chloroform to the sample. Homogenize for another 2 min. Flush with argon gas. Keep the sample in an ice bucket for as much as possible.
i) Optional: vortex for 30 s.
j) Flush samples in argon gas before centrifugation.
k) Centrifuge samples at 13,000 RPM (11,337 × g) for 15 min. When centrifugation is completed, there should be three layers: the superior aqueous layer, the middle tissue layer, and the inferior organic layer. The lipids are contained in the organic layer.
l) In four separate tubes, evenly split the organic layer. For the solution above, there is a total of 550 μL of chloroform, so add 135 μL of the organic layer in each of the tubes. These are the lipid aliquots. Because ocular tissue has a low lipid yield, four aliquots of trabecular meshwork lipids allow for detectable amounts, and keep mass spectrometer clean from excess lipids. For brain, liver, or other tissues where lipid yield is expected to be high, we recommend using either the Folch or MtBE method of lipid extraction and increasing the number of aliquots as keep the mass spectrometer analyses clean as possible.
m) Flush each aliquot with argon gas.
n) Speed-vac with no heat to vaporize the remaining chloroform in the organic layer, until lipid samples are completely dry.
o) Flush with argon gas.
p) Store at −80 °C until mass spectrometric analysis.
3 Protein Quantification
a) Speed-vac the tubes with the sample containing the aqueous layer and the remaining tissue until the tissue is barely moist. You may use the temperature up to 30 °C if desired, however, be careful that the tissue is not burned from staying too long in the heated Speed-Vac.
b) Store at −80 °C until ready for protein quantification.
c) Use 0.05% SDS as a buffer for protein extraction. To make: Add 25 μL of sodium dodecyl sulfate to 49.975 mL of HPLC water for a 0.05% SDS solution.
d) Keep tubes with samples in ice bucket as much as possible in the protein extraction phase.
e) Add 400 μL of 0.05% SDS buffer to samples with tissue.
f) Homogenize with handheld homogenizer with tips sterilized in 70% ethanol for 2 min.
g) Vortex for 2 min.
h) Centrifuge for 13,000 RPM (11,337 × g) for 15 min.
i) Separate the supernatant or the top aqueous phase containing the proteins into different tubes.
j) Speed-vac to less than 50 μL with optional heat up to 30 °C. Because ocular tissues are small and have a low protein yield when compared to other common biological samples, our protein samples needed to be concentrated further, down to 15–20 μL at times for protein quantification to be done correctly.
k) Add 0.5 μL of BSA and add to 999.5 μL of HPLC water to create a 0.1 μg/μL BSA standard solution.
l) Take a 96 ELISA well-plate and label the wells appropriately with at least three separate readings per sample or standard.
m) Dilute the protein reagent assay 1:10 with distilled water. We make 50 mL of protein reagent solution stock at a time, and store in the 4 °C freezer by diluting 5 mL of reagent with 45 mL of distilled water.
n) By Bradford method, a protein standard should encompass concentrations from 0 to above the highest protein concentration contained in one of the samples to successfully extrapolate the protein concentration of samples. Because if the low protein concentration is expected in ocular tissues, we use 0, 1, 2, 4, 6, and 8 μg/μL BSA concentrations as a standard.
o) To make the 1 μg/μL standard: add 0.4 μL of BSA standard into 399.6 μL of diluted protein reagent in a tube. This solution now has a concentration of 1 μg/μL.
For the 2 μg/μL standard: add 0.8 μL of BSA standard into 399.2 μL of the protein reagent.
For the 4 μg/μL standard: add 1.6 μL of BSA standard into 398.4 μL of the protein reagent.
For the 6 μg/ μL standard: add 2.4 μL of BSA standard into 397.6 μL of protein reagent.
For the 8 μg/μL standard: add 3.2 μL of BSA standard into 396.8 μL of protein reagent in a tube.
p) Add to each of the three wells designated as the 0 μg/μL, 100 μL of the protein reagent alone.
q) Add to three wells, 100 μL of the 1 μg/μL standard each. Add to three more wells, 100 μL of the 2 μg/μL standard each. Repeat for the 4 μg/μL standard, the 6 μg/μL standard, and the 8 μg/μL standard.
r) Add 99 μL of the protein assay reagent into three wells. Add 1 μL of the protein sample (the supernatant that has been concentrated by speed-vac and stored at −80 °C) to each of the three wells for a total volume of 100 μL.
s) Repeat step 18 for each of the samples.
t) Repeating the absorbance three times will provide maximum accuracy and precision of both the standard and the subsequent protein concentration of the samples.
u) Obtain the absorbance using instrumentation available.
v) Create a curve with the concentration of the standards on the x-axis and the corresponding absorbance on the y-axis.
w) Calculate the concentrations of the protein samples.
x) Multiply the concentrations in μg/μL by the total number of μL for each supernatant. This is the total μg of protein in each sample, and the normalization for the total amount of lipids obtained from the mass spectrometric analysis, i.e., lipid amounts are expressed as pmol of lipid/μg protein.
y) For even lower protein amounts unable to detect using a Bradford method, we recommend the use of the PHAST gel densitometry with a BSA standard for protein quantification.
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Reference
- Bhattacharya, S. K. (2017). Lipidomics. Methods in Molecular Biology, 1609.
