TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

The major benefits of this protocol are that it reduces the costs of labeling, improves protein extraction, and generates high-quality data. The major advantages of our protocol are the high labeling efficiencies for the different types of samples, its reproducibility, and the reliable data acquisition. After isolating quadriceps muscle tissue samples from a euthanized mouse according to an IACUC approved protocol, weigh out 10 milligrams of muscle tissue and use tweezers to separate the muscle fibers.

To create a lysate, transfer the separated tissue sample into a two milliliter tube containing 200 microliters of one millimeter zirconia silica beads and 500 microliters of CHAPS lysis buffer. Place the samples in a bead disruptor and perform two sequential 45-second runs. After the second cycle, to release the DNA-bound protein, sonicate the sample 10 times for 10 seconds per sonication at a 50%amplitude with 30-second intervals on ice.

After the last sonication, centrifuge the lysate and transfer the supernatant to a new centrifuge tube. For reducing/alkylating reagent treatment, transfer 200 micrograms of protein per condition into new centrifuge tubes and use fresh CHAPS lysis buffer to bring the final volume in each tube up to 100 microliters. Next, add five microliters of TCEP to each tube and incubate the samples at 55 degrees Celsius for one hour.

At the end of the incubation, dissolve nine milligrams of iodoacetamide in 132 microliters of 100 millimolar TEAB and protect the resulting 375 millimolar iodoacetamide solution from light. Then add five microliters of iodoacetamide to each sample and incubate for 30 minutes at room temperature protected from light. For methanol/chloroform precipitation, add 400 microliters of methanol to each 100 microliters of protein and briefly vortex the samples.

To incorporate the liquids deposited on the sides of the sample tubes, briefly centrifuge the samples and add 100 microliters of chloroform to each tube. After a brief vortexing, quickly centrifuge the tubes again to collect the liquid on the sides of the tubes and add 300 microliters of water to the samples. Vortex vigorously to obtain homogenous solutions and centrifuge the samples for one minute under the same centrifuge conditions.

At the end of the centrifuge, transfer the tubes carefully to a rack without disturbing the layers and carefully remove the supernatant. Add 300 microliters of methanol to the remaining inter and bottom phases and vortex the samples vigorously again. Centrifuge the samples for two minutes and carefully aspirate the supernatants.

Then gently aspirate as much liquid from each sample as possible under a stream of air until the pellets are just a bit moist. Assess the hydration every two minutes for about 10 minutes. If needed, the pellets can be stored at minus 80 degrees Celsius until further processing.

To digest the protein, resuspend the precipitated pellet in 100 microliters of TEAB lysis buffer and add 100 microliters of trypsin storage solution to the bottom of a 100 microgram trypsin glass vial. After five minutes at room temperature, add 2.5 microliters of the freshly prepared trypsin solution per 100 micrograms of protein to each sample and incubate the samples at 37 degrees Celsius overnight. For peptide labeling, dissolve each 0.8 milligram TMT tag vial with 41 microliters of anhydrous acetonitrile per tag and incubate the reagents for five minutes at room temperature with occasional vortexing while keeping the labels protected from light.

At the end of the incubation, briefly centrifuge the vials and carefully add 41 microliters of TMT label reagent to each 100 microliter sample for a one-hour incubation at room temperature. At the end of the incubation, quench the reactions with the addition of eight microliters of 5%hydroxylamine for 15 minutes at room temperature. Then split the samples into equal aliquots in new centrifuge tubes for storage at minus 80 degrees Celsius.

In this representative data acquisition, 39, 653 total peptides were analyzed, of which 4, 457 had equal or greater than two unique peptides and 3, 829 included reporter ions for all of the channels. A fold change cutoff was used to determine the relative distribution of proteins from diseased compared to healthy cells with 296 proteins of significantly lower abundance and 108 proteins of significantly higher abundance observed. PANTHER analysis showed a categorized list of proteins based upon significantly lower or higher abundance of diseased cells based upon their molecular function.

String analysis of the proteins of significantly lower or higher abundance further identified multiple interactions and strong associations between proteins. Additional methods can be performed to identify protein-to-protein interactions, to classify the interactions by groups, and to identify the interactions in a variety of pathways. Proteomics techniques are powerful tools within the biomedical research fields for understanding the mechanisms of disease development by providing detailed protein abundance information.