TITLE: Tissue Sample Preparation for LC-MS Analysis
AUTHORS: Joanne   Chan, Ruiqi Jian, Lihua Jiang

[DESCRIPTION]
This protocol describes the procedure for protein extraction, enzymatic digestion, sample cleanup an dTMT labeling for LC-MS analysis. Proper sample preparation and clean-up are extremely important to ensuring quality LC-MS data and reproducibility. Tissues tend to vary more than cells and extra care should be taken as MS is sensitive to any sample variations. In addition, proper clean-up is a preventative measure to less damage to the instruments and superior data. Here, we described each step in detail on how to process tissue samples prior to analysis.

[BEFORE_START]
Ensure you have enough tissue before preparation. We recommend using at least 30 mg of tissue to start with.

[GUIDELINES]
Please make sure at any point in the experiment, do not leave the samples unattended. If not actively performing a step, put your samples on ice.

[STEPS]
SECTION: Protein Lysate Extraction
1. Place the tissue chunk onto a cell culture plate on ice and mince with disposable scalpels to the best of your abilities.
SECTION: Protein Lysate Extraction
2. Add 500 µL  lysis buffer (6 Molarity (M) GdmCl, 10 Milimolar (mM) TCEP, 40 Milimolar (mM) CAA, 100 Milimolar (mM) Tris pH 8.5) to the minced tissue sample and transfer it to a Matrix D tube with ceramic beads. 

SECTION: Protein Lysate Extraction
3. Beat the tissue using FastPrep-24™ at 4.5 M/S, 40 s at 4 °C 2 times .

SECTION: Protein Lysate Extraction
4. Centrifuge the sample for 5 min  at 7000 x g, on ice , and transfer the sample to a new tube, and sonicating at 40% and 60% for 20 s each, respectively on ice.

SECTION: Protein Lysate Extraction
5. Heat for 5 min  at 95 °C and vortex every 1 min .

SECTION: Protein Lysate Extraction
6. Centrifuge for 5 min , 10 min , and 10 min minutes respectively at 12000 x g, and collect the supernatant at each step.

SECTION: Enzymatic Digestion
7. Take 5 µL of the sample out and dilute to measure its protein concentration using BCA kit.

SECTION: Enzymatic Digestion
8. Take 100 µg -300 µg  of the sample to perform a two-step enzymatic digestion using 25 Milimolar (mM) Tris pH 8.5 as the dilution buffer.
SECTION: Enzymatic Digestion
9. Endoproteinase Lys-C digestion: Add 3x volume of dilution buffer to the original lysate. Add Lys-C to lysate at 1:100 (w:w) and incubate at 37 °C for 120 min.

SECTION: Enzymatic Digestion
10. Trypsin digestion: Add an additional 6x volume of dilution buffer to the original lysate volume for an overall 9x dilution of the original sample volume. Add Trypsin to lysate at 1:50 (w:w) and incubate at 37 °C   .

SECTION: Enzymatic Digestion
11. Digested samples can be stored at -80 °C .

SECTION: Desalting the Peptide
12. Desalt the digest with Waters Oasis® HLB Cartridge.
SECTION: Desalting the Peptide
12.1. Condition the column with 1 mL each of  100 % (v/v) acetonitrile, 80 % (v/v) acetonitrile with 0.1 % (v/v) acetic acid, and 0.1 % (v/v) acetic acid sequencially.
SECTION: Desalting the Peptide
12.2. Adjust the sample to 1 % (v/v) TFA, and load the sample onto the desalting column.
SECTION: Desalting the Peptide
12.3. Wash the sample with 200 µL of 0.1 % (v/v) acetic acid 3 times.
SECTION: Desalting the Peptide
12.4. Elute peptides off the column with 150uL of 40 % (v/v) acetonitrile with 0.1 % (v/v) acetic acid, followed by 150uL of 80 % (v/v) acetonitrile with 0.1 % (v/v) acetic acid.
SECTION: Desalting the Peptide
13. Put the sample in the SpeedVac until dry.
SECTION: TMT Labeling
14. The TMT10plex™ Isobaric Label Reagents were prepared as suggested by the manufacture.   https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0016969_2162457_TMT10plex_UG.pdf&title=VXNlciBHdWlkZTogVE1UMTBwbGV4IE1hc3MgVGFnIExhYmVsaW5nIEtpdHMgYW5kIFJlYWdlbnRz
SECTION: TMT Labeling
14.1. Resuspend the sample in 100 Milimolar (mM) TEAB pH 8.5 and then add assigned TMT tag to the sample. 

SECTION: TMT Labeling
14.2. Incubate the sample with its associated TMT tag for 60 min at room temperature.

SECTION: TMT Labeling
14.3. Quench the reaction with 5 % (v/v) hydroxylamine.

SECTION: TMT Labeling
14.4. Mix each set of 10 samples together in equal amounts.
SECTION: TMT Labeling
15. Put the sample in the SpeedVac to dry.
SECTION: TMT Labeling
16. Resuspend the sample with 200 Milimolar (mM) ammonium formate and perform LC-MS analysis.