Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Our automated cPILOT protocol makes it easier for researchers to analyze samples in a high throughput way. This is significant because it allows us to get to biological information about diseases or different conditions a lot quicker. The main advantage of this technique is that it helps to process multiple samples in parallel, thereby reducing actionable errors while using high throughput samples.

Our laboratory is interested in applications related to aging and Alzheimer's disease. However, this technique can also be used to study any disease or challenge for where there are biological tissues involved. Begin by turning on the positive pressure apparatus, the heating and cooling device, and the vacuum pumps.

Connect all accessories with the liquid handler, which will show a blue light once that is connected to the computer and ready to operate. Aliquot 300 microliters of the liver homogenate into a 500 microliter tube and place it on a two milliliter deep well plate. Store the sample at four degrees Celsius until the start of the protocol.

Open the method in the software and place the required tips and labware into their positions. Then crosscheck with the final deck layout and click Next to continue the protocol. Load 230 microliter tips and aspirate 90 microliters of eight molar urea from the reservoir.

Then dispense it to row one of the black two milliliter deep well plate. Unload the tips of TL1 and repeat this step until urea has been added to all the wells. After adding the denaturation buffer, use 90 microliter tips to transfer 10 microliters of mouse liver homogenate to the deep well plate.

Unload the tips, then load one row of 90 microliter tips and aspirate three microliters of DTT from reagent plate one. Dispense DTT to rows one and two of the deep well plate. Seal the sample plate with aluminum foil and incubate it at 37 degrees Celsius for 600 seconds while rotating at 300 RPM.

After the incubation, load one row of 90 microliter tips, aspirate six microliters of IAM from reagent plate one at row three, and dispense it to row one of the sample plate. Seal the sample plate and incubate it at four degrees Celsius for 30 minutes while rotating at 300 RPM on the cooling device. Then unseal the sample plate and load one row of 90 microliter tips.

Aspirate five microliters of cystine from reagent plate one at row two and dispense it to rows one and two of the sample plate. Incubate the plate at room temperature for 30 minutes. After the incubation, perform a timed shake of 1800 RPM for 30 minutes.

Then add 800 microliters of 20 millimolar Tris buffer with 10 millimolar calcium chloride to each well on the sample plate to dilute the urea concentration to two molar. Add 20 microliters of trypsin to the first row of a 96 well plate and place the plate at a specified location on the deck. After adding the trypsin to the sample plate, seal it and incubate it for 15 hours at 37 degrees Celsius and 600 RPM on the heating and cooling device.

Stop the digestion by aspirating 150 microliters of 5%formic acid from row three of the formic acid plate and dispensing it to the sample plate at rows one and two. Proceed to desalt the peptides. Use 1070 microliter tips to aspirate 600 microliters of acetonitrile.

Then dispense it in rows one and two of the solid phase extraction, or SPE plate, to activate C-18. Next, load the digested sample to the SPE plate in two passes. Load two rows of tips, aspirate 534 microliters of the digested samples, and dispense them to rows one and two of the SPE plate.

Apply pressure to the plate and repeat this step until all the samples are loaded. Clean the peptides by washing with water and dilute the peptides with 60 to 40 acetonitrile in 0.1%formic acid. Then place the plate on top of a collection plate to elute the peptides using the positive pressure apparatus.

Draw down the flow-through and set up the required tips and labware in the software, crosscheck with the deck layout, and click Next to continue the protocol for dimethylation. Reconstitute the peptides in 1%acetic acid. To perform dimethylation labeling, load 90 microliter tips and aspirate 16 microliters of 60 millimolar light formaldehyde from the second row of reagent plate two.

Dispense it to row one of the sample plate, then unload the tips. Load one row of 90 microliter tips and aspirate 16 microliters of 60 millimolar heavy formaldehyde from row three of reagent plate two. Dispense it row two of the sample plate and unload the tips.

Load two rows of 90 microliter tips and aspirate 16 microliters of 24 millimolar sodium cyanoborohydride and 24 millimolar sodium cyanoborodeuteride from rows one and two of reagent plate three, then dispense the reagents to rows one and two of the sample plate to start the dimethylation reaction. Unload the tips and use the orbital shaker to perform a timed shake for 15 minutes at 1800 RPM. Then load two rows of 90 microliter tips and aspirate 32 microliters of 1%ammonia from rows three and four of reagent plate three.

Dispense it into rows one and two of sample plate two to stop the reaction. Combine equal volumes of light and heavy dimethylated peptides in a new two milliliter deep well plate for desalting. After desalting the combined light and heavy peptides, dry down the peptides and perform isobaric tagging.

Reconstitute the peptides with 100 microliters of 100 millimolar triethylmonium bicarbonate buffer on an orbital shaker. Then use 90 microliter tips to aspirate 25 microliters of the combined dimethylated peptides and dispense them to the first row of the TMT processing plate. Load one row of 90 microliter tips and aspirate 10 microliters of TMT.

Dispense it in the first row of the TMT processing plate, unload the tips, then perform a timed shake for one hour at 1800 RPM. Aspirate eight microliters of hydroxylamine from row two of the TEAB plate and dispense it to row one of the TMT processing plate. After a 15 minute shake at 1800 RPM, transfer 30.5 microliters of the TMT labeled peptides to a 1.5 milliliter tube.

Then proceed with data acquisition and analysis according to the manuscript directions. Representative MS data of a peptide identified in all 22 reporter ion channels from a 22 Plex combined precursor isotopic labeling and isobaric tagging experiment is shown here. The sequence of the peptide corresponds to betaine-homocysteine S-methyltransferase.

The most intense fragment ions for both the light and heavy dimethylated peaks were further isolated for MS3 fragmentation. The reporter ion intensities are directly proportionate to the peptide abundance in the sample. Overall, this combined precursor isotopic labeling and isobaric tagging experiment reduced the sample processing times and made it possible to visualize the protein expression across multiple channels or conditions.

The box plot of log 10 abundance versus total reporter ion intensities across all the 22 channels shows lesser inter-well or inter-sample variability. Evaluation of the total automation was done by examining the error in reporter ion abundance across each protein in the 22 samples. Sample processing with the robotic platform resulted in very low coefficients of variation.

Across the 3098 peptides, the average coefficient of variation in reporter ion abundance was 12.36 and 15.03%for light and heavy dimethylated peptides respectively. When attempting this protocol, it is important to keep in mind that isotopic and isobaric levels are used in a single experiment. Hence, careful planning needs to be performed to designate each sample with the tags intended to be used in the experiment.

This method can be readily integrated into other robotic systems, and it can be applied to a variety of tissues, such as tissue or cell lysates and other biofluids.