Tissue Sample Preparation for LC-MS Analysis
Joanne Chan, Ruiqi Jian, Lihua Jiang
HuBMAP
BIOMIC
MSRC
Stanford
Proteomics
Peptides
Protein Precipitation
Desalting
Tissue Lysis
Protein Digestion
TMT labeling
Abstract
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.
Steps
Protein Lysate Extraction
Place the tissue chunk onto a cell culture plate on ice and mince with disposable scalpels to the best of your abilities.
Add 500µL lysis buffer (6Molarity (M) GdmCl, 10millimolar (mM) TCEP, 40millimolar (mM) CAA, 100millimolar (mM) Tris 8.5) to the minced tissue sample and transfer it to a Matrix D tube with ceramic beads.
Beat the tissue using FastPrep-24™ at 4.5 M/S, 0h 0m 40s at 4°C 2 times .
Centrifuge the sample for 0h 5m 0s at 7000x g,0h 0m 0s , and transfer the sample to a new tube, and sonicating at 40% and 60% for 0h 0m 20s each, respectively on ice.
Heat for 0h 5m 0s at 95°C and vortex every 0h 1m 0s .
Centrifuge for 0h 5m 0s , 0h 10m 0s , and 0h 10m 0s minutes respectively at 12000x g,0h 0m 0s, and collect the supernatant at each step.
Enzymatic Digestion
Take 5µL of the sample out and dilute to measure its protein concentration using BCA kit.
Take 100µg -300µg of the sample to perform a two-step enzymatic digestion using 25millimolar (mM) Tris 8.5 as the dilution buffer.
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 2h 0m 0s.
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 .
Digested samples can be stored at -80°C .
Desalting the Peptide
Desalt the digest with Waters Oasis® HLB Cartridge.
Condition the column with 1mL 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.
Adjust the sample to 1% (v/v) TFA, and load the sample onto the desalting column.
Wash the sample with 200µL of 0.1% (v/v) acetic acid 3 times.
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.
Put the sample in the SpeedVac until dry.
TMT Labeling
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
Resuspend the sample in 100millimolar (mM) TEAB 8.5 and then add assigned TMT tag to the sample.
Incubate the sample with its associated TMT tag for 1h 0m 0s at room temperature.
Quench the reaction with 5% (v/v) hydroxylamine.
Mix each set of 10 samples together in equal amounts.
Put the sample in the SpeedVac to dry.
Resuspend the sample with 200millimolar (mM) ammonium formate and perform LC-MS analysis.
