The presented protocol allows to quantify a broad range of lipid in animal tissue. It's an easy tool to study lipid mechanisms and also to identify biomarker indicating early toxicity in animal models. The method delivers a quantitative profile of more than 400 molecular lipids in 14 different lipid classes in a broad variety of sample matrices with a 10-minute trend time per sample.
To begin, dilute centrifuge aliquot 1 supernatant twice the concentration with ammonium bicarbonate buffer. After preparing the bovine serum albumin standard curve, vortex the standard tubes and centrifuge the tubes at 18, 200 g for 15 seconds at room temperature. From the standard tubes prepared previously, transfer 6 microliters each of the blank, standards, and samples to a 96-well flat-bottom plate.
Next, add 250 microliters of the Bradford reagent to each well by using a multi-channel pipette and mix. After incubating for 5 minutes, measure the absorbance at a 595 nanometer wavelength by using a plate reader. Prepare 2 milliliter tubes with 100 microliters of ammonium bicarbonate solution into each tube of the total blank and internal standard blank and keep all samples on ice.
Prepare quality control samples, considering one quality control sample is placed between each set of 10 samples. Add 10 microliters of pooled human plasma to 2 milliliter tubes, then spike 10 microliters of internal standard solution into all internal standard blank, quality control, and study samples. Add 300 microliters of minus 20 degrees Celsius butanol methanol mixture to each sample and shake at 450 g for 10 minutes at room temperature on thermomixer.
Next, add 300 microliters of heptane ethyl acetate mixture and shake at 450 g for 10 minutes at room temperature on the thermomixer, then add 300 microliters of 1%ascetic acid mixture and shake for 5 minutes at 450 g at room temperature on the thermomixer. Centrifuge at 2, 800 g for 5 minutes at room temperature and transfer 360 microliters of the upper phase to a new 2-milliliter self-lock tube labeled as 2. After adding 320 microliters of heptane ethyl acetate to the water phase tube labeled as 1, shake at 450 g for 5 minutes at room temperature on the thermomixer, then centrifuge at 2, 800 g for 5 minutes at room temperature as demonstrated previously.
Transfer 320 microliters of the upper phase and combine them with the fraction from the previous step in tube 2. Similarly, add 250 microliters of heptane ethyl acetate to the water phase in tube 1 and shake at 450 g for 5 minutes at room temperature on the thermomixer. Again, centrifuge at 2, 800 g for 5 minutes at room temperature, then transfer 200 microliters of the upper phase and combine it with the fractions from the previous steps in tube 2.
Evaporate to dryness at 35 degrees Celsius in a vacuum concentrator and re-dissolve in 300 microliters of MS mix solution. Vortex each tube for 5 seconds to ensure that everything is dissolved and centrifuge at 18, 200 g for 5 minutes at 4 degrees Celsius. Transfer an aliquot of 50 microliters into the microliter plate for positive ionization mode analysis and dilute with 50 microliters of MS mix solution.
Again, dilute with 80 microliters of MS mix and transfer an aliquot of 20 microliters into the MTP plate for negative ionization mode analysis. Wrap the plate with foil and keep at 4 degrees Celsius before the analysis. Calibrate an MS both in positive and negative modes in accordance with the manufacturer's instructions 5 days or less before the analysis.
Install the direct infusion nano-source upfront, ensuring that it's properly aligned against the transfer capillary of the MS and a 4.1 micrometer nozzle chip into the chip holder, then check the parameters for positive and negative infusion corresponding to a gas pressure of 1.25 psi, source voltage of 1.1 kilovolt, 5 microliters of sample injection volume, output contact closure Rel1 for 2.5 seconds, 5 minutes of delivery time, plate cooling at 4 degrees Celsius, then set up the temperature controller and create a new plate. For positive mode, check and tune software that the MS method is set up with a capillary temperature of 250 degrees Celsius and the S Lens RF level at 65.0. In Xcalibur software, open the positive method and check the full-scan MS acquisition is set up from zero to 1 minute in the 550 to 1, 000 m/z range at 140, 000 resolution with automated gain control of 1 million and maximum injection time of 50 milliseconds.
Apply lock mass of 680.48022. Check that the data-independent MS/MS acquisition method is set up between the 1 and 5-minute time range at 17, 500 resolution with a fixed first mass of 250 m/z. Check that the automated gain control for MS2 is set up at 100, 000 and a maximum injection time at 64 milliseconds, a collision energy of 20 NCE, the isolation window at 1 m/z and that an inclusion mass list from 398 to 1, 100 m/z is used with a mass step of 1 Dalton.
For the acquisition in negative mode, check in tune software that the MS method is set up with the capillary temperature at 250 degrees Celsius in the S-lens RF level at 65.0 in the MS tune file. Next, in Xcalibur software, check the full-scan acquisition mode is set up from zero to 1 minute at 140, 000 resolution covering the 400 to 940 m/z range, automated gain control of 1 million, maximum injection time of 200 milliseconds, and a lock mass of 526.46262. Check that the MS2 acquisition in DIA mode is set up between 1 and 5 minutes of the run at a resolution of 17, 500 with a fixed first mass of 150 m/z, automated gain control of 100, 000, 64 milliseconds of maximum injection time and 35 NCE of collision energy using the inclusion mass list from 400 to 940 with a mass step of 1 Dalton.
Create the analysis sequence queue in Xcalibur software in positive mode and trigger it, then create the sequence in ChipSoft software and start the analysis while waiting for the sample acquisition to be triggered by a contact closure signal coming from the direct infusion robot for each sample. When positive mode analysis is complete, create the analysis sequence queue in Xcalibur software in negative mode and trigger it, then create the sequence in ChipSoft software and start the analysis while waiting for the sample acquisition to be triggered by a contact closure signal coming from the direct infusion robot for each sample. The total normalized lipid concentration was identified and quantified from the 14 most abundant lipid classes, which was slightly elevated for the PE, PEO, PC, PCO, PI, and PG lipid classes and some down regulation was observed for SM, LPE, and PA lipids.
While no difference could be seen for TAGs and PS.These results also show that, among the molecular features based on the total molecular formula, 100 to 120 compounds were significantly impacted by cigarette smoke exposure. The deconvolution of the MS2 fragmentation and normalization of the fragment signal intensities allowed the approximate definition of the total amount of each conjugated fatty acid represented in each lipid glass and comparison of these data between exposed and control groups. A decrease in fully saturated and few unsaturated fatty acids was observed, whereas the polyunsaturated fatty acids in the composition of PC, PE, and PG phospholipid classes were elevated in the cigarette smoke group for all time points.
The extreme cases of PC and PG conjugated with eicosapentaenoic and docosahexaenoic acid were exclusively detected in the 3R4F CS exposure group. Pipetting of the sample and QC sample has to be done with low-band tips and to be accurate, meaning the tips has to be checked between each samples. The pipetting of the internal standard solution is critical and has to be accurate.
The solution of 1 to 1 dichloromethane methanol has to be handled with care and a non-plasticizer containing tips has to be used to avoid having plastic polymers in the samples. The tips must be changed between each samples.
