Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

This protocol uses automation to perform protein biochemistry preparations prior to mass spectrometry experiments, allowing higher throughput and lower variability for proteomic studies. This protocol uses a low-cost, programmable liquid handling system that is affordable to more laboratories. We provide open source Python scripts that can be modified for further development.

Demonstrating the procedure will be Milton Amaya, a master's student in our laboratory. To begin, open the noSP3_Digestion. py script in the text editor and specify the experiment-specific variables as needed in the CUSTOMIZE HERE ONLY section.

Then, open the Opentrons app and upload the script to the Protocol tab in the Opentrons app. Next, place the required labware and pipettes in the corresponding location in the OT2 deck specified in the Python script. Then, place the ammonium bicarbonate solution in the A1 well of the four-in-one tube rack with the 15-milliliter plus 50-milliliter tube holder top and place the protein sample in the A1 well of the four-in-one tube rack with a two-milliliter tube holder top.

Manually place two-milliliter protein low-bind tubes in the wells of the aluminum block placed on top of the temperature module, starting from A1 and moving down vertically. Then, place the DTT solution in the A6 well of the four-in-one tube rack with a two-milliliter tube holder top. Observe while the robot transfers an appropriate volume of the ammonium bicarbonate buffer to the sample tubes in the aluminum block.

Then, manually verify that the robot program is paused and displays the message, Ensure DTT has been loaded into A6 of the two-milliliter tube rack located in slot four before resuming protocol. After confirming the location of the DTT tube and opening the tube cap, click on the Resume button in the Opentrons app to continue. Ensure that the robot transfers 10 microliters of the DTT solution to each sample well, followed by five mixing rounds.

Next, verify that the robot program is paused and displays the message, Ensure to close caps on sample tubes. Then, manually close the caps of the tubes and click on Resume to continue. Wait till the robot's temperature module starts to heat the aluminum block and the temperature reaches 55 degrees Celsius, followed by a five-minute incubation to allow the samples to get to 55 degrees Celsius.

The robot will then hold this temperature for 30 minutes to allow protein reduction by DTT. During the incubation, prepare iodoacetamide solution and manually wrap it with aluminum foil to avoid exposure to light. After the incubation, when the robot's program is paused and displays the warning message, Ensure to open caps on sample tubes, uncap the sample tubes and click on Resume to continue.

Next, manually verify that the robot's program is paused with the warning message, Ensure iodoacetamide has been loaded into B6 of the two-milliliter tube rack located in slot four before resuming protocol. After confirming the rack location of the iodoacetamide tube and opening the tube cap, click on Resume and ensure that the robot transfers 10 microliters of the iodoacetamide solution to each sample tube, followed by five mixing rounds. When the robot's program is paused and displays the message, Close caps on sample tubes, cap the sample tubes and cover them with foil, then cover the entire aluminum block with a clean piece of foil and click on Resume to continue.

Wait till the samples are incubated at 22 degrees Celsius for 30 minutes, and ensure that the robot's temperature module deactivates upon completion of the iodoacetamide incubation. When the robot's program is paused and displays the warning message, Ensure trypsin has been loaded into C6 of the two-milliliter tube rack located in slot four prior to resuming protocol, place trypsin solution in the C6 well of the two-milliliter tube rack with the tube cap open, and click on Resume to continue. Then, when the robot's program is paused and displays the warning message, Open caps on sample tubes on the temperature module, uncap the sample tubes and click on Resume to continue.

Standby while the robot transfers 10 microliters of trypsin to each sample tube, followed by five mixing rounds. After overnight trypsin digestion, briefly spin the samples using a benchtop microcentrifuge and place the samples on a magnetic tube rack. After two minutes, transfer the supernatant carefully with a pipette to a new set of protein low-bind microcentrifuge tubes, and keep the samples in a refrigerator.

Next, open the SP3_peptide_cleanup. py Python script in a text editor, and specify the experiment variables as needed in the CUSTOMIZE HERE ONLY section. Then, upload the script to the Protocol tab in the Opentrons app.

Place the required labware and pipettes in the corresponding location in the OT2 deck specified in the Python script, and make sure that the magnetic module is powered on and connected to the robot. Place a new, two-milliliter, 96-well deep well plate on the top of the magnetic module. Next, place the digested samples in the two-milliliter tube rack starting from A1 and moving down vertically.

Then, ensure of the robot transfers 55 microliters of the digested samples to the wells in the deep well plates on the magnetic module. Verify that the robot protocol is paused and displays the message, Ensure prepared beads have been loaded into A6 of the two-milliliter tube rack located in slot four before resuming protocol. Then, vortex and briefly spin the SP3 beads in a mini-benchtop centrifuge and place them in the A6 well of the two-milliliter tube rack with the cap open.

Verify that the robots program is paused and displays the message, Ensure 80%ethanol has been loaded into A4 of the 15-milliliter plus 50-milliliter tube rack located in slot five prior to resuming protocol. Then, place an 80%ethanol solution in the A4 well in the tube rack and click on Resume to continue. Observe while the robot changes the pipetting speed to slow, aspirates the supernatant from each well, and dispenses it into the waste tube.

Wait till the robot changes the pipetting speed back to default and disengages the magnetic module. When the robots program is paused and displays the message, Open cap on ammonium bicarbonate tube, open the cap of the ammonium bicarbonate solution, then click on Resume to continue. Standby while the robot disengages the magnetic module, transfers 250 microliters of the ammonium bicarbonate buffer to each well, and immediately mixes for 10 times.

Then, observe while the robot changes the pipetting speed to slow, and transfers the supernatant from each well to the waste tube. Wait till the robot transfers 100 microliters of the ammonium bicarbonate buffer to each well and immediately mixes for 10 times. Verify that the robots program is paused and displays the message, Ensure new collection tubes have been placed in the two-milliliter aluminum block prior to resuming protocol.

Then, place a new set of low-protein-retention microcentrifuge tubes in the aluminum block immediately after the last sample tube initially in the block, and click on Resume to continue. Standby while the robot transfers each sample in the ammonium bicarbonate buffer to the new two-milliliter tubes. When the robot's program is paused and displays the message, Ensure trypsin has been loaded into C6 of the two-milliliter tube rack located in slot four prior to resuming protocol, place the trypsin solution in the C6 well of the two-milliliter tube rack with the tube cap open, and click on Resume to continue.

Once the program is finished running, wrap the sample tube caps with paraffin film, transfer all the samples to a temperature-controlled mixer, and incubate at 37 degrees Celsius for 16 to 20 hours with 1, 000 RPM shaking. With the BSA sample, a medium of 728 peptide spectrum matches and 65 peptides were identified, with 5.2%and 3.2%coefficients of variation, respectively. With a complex heart sample, a medium of 9, 526 peptide spectrum matches, 7, 558 peptides, and 1, 336 proteins was identified in 10 runs with 7.6%5.9%and 3.6%coefficients of variation, respectively.

To determine the variability in peptide quantification, the coefficients of variation of the extracted ion chromatogram intensities were calculated for 10 peptides that map to a unique protein. When the variabilities of human versus robot experimental results in measuring protein concentration were further compared with the BCA assay, the average coefficient of variation of the robot BCA assay was lower than the human manual BCA assay. This protocol reduces the bench time for processing each sample.

It paves the way for us to explore proteomics differences in a panel of cell lines in between multiple drug treatments.