Apr 06, 2022

Public workspaceTissue Sample Preparation for LC-MS Analysis

DOI
dx.doi.org/10.17504/protocols.io.bgf5jtq6
  • 1Stanford University
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
  • dGTEX protocols
Joanne Chan
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DOI: dx.doi.org/10.17504/protocols.io.bgf5jtq6
Protocol Citation: Joanne Chan, Ruiqi Jian, Lihua Jiang 2022. Tissue Sample Preparation for LC-MS Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bgf5jtq6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 15, 2020
Last Modified: April 06, 2022
Protocol Integer ID: 37085
Keywords: 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.
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.
Materials
MATERIALS
ReagentLysing matrix D tubesMP BiomedicalsCatalog #6913100
Reagent2-Chloroacetamide (CAA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #22790
ReagentTrizma® baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #93350
ReagentAcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #179124
ReagentGuanidine-HClThermo FisherCatalog #24110
ReagentPierce™ Trypsin Protease, MS GradeThermo FisherCatalog #90059
ReagentTMT10plex™ Isobaric Label Reagent Set, 1 x 0.8 mgThermo FisherCatalog #90110
ReagentTris(2-carboxyethyl)phosphine hydrochloride (TCEP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #C4706
ReagentLysyl Endopeptidase Mass Spectrometry Grade (Lys-C)FUJIFILM Wako Pure Chemical CorporationCatalog #12505061
ReagentOasis HLB 1 cc Vac Cartridge 10 mg Sorbent per Cartridge 30 µm 100/pkWatersCatalog #186000383
Safety warnings
All the waste needs to go to biohazard.
Before start
Ensure you have enough tissue before preparation. We recommend using at least 30 mg of tissue to start with.
Protein Lysate Extraction
Protein Lysate Extraction
4h
4h
Place the tissue chunk onto a cell culture plate on ice and mince with disposable scalpels to the best of your abilities.
Add Amount500 µL lysis buffer (Concentration6 Molarity (M) GdmCl, Concentration10 millimolar (mM) TCEP, Concentration40 millimolar (mM) CAA, Concentration100 millimolar (mM) Tris Ph8.5 ) 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, Duration00:00:40 at Temperature4 °C 2 times .

Centrifuge the sample for Duration00:05:00 at Centrifigation7000 x g , and transfer the sample to a new tube, and sonicating at 40% and 60% for Duration00:00:20 each, respectively on ice.

Heat for Duration00:05:00 at Temperature95 °C and vortex every Duration00:01:00 .

Centrifuge for Duration00:05:00 , Duration00:10:00 , and Duration00:10:00 minutes respectively at Centrifigation12000 x g , and collect the supernatant at each step.

Enzymatic Digestion
Enzymatic Digestion
16h
16h
Take Amount5 µL of the sample out and dilute to measure its protein concentration using BCA kit.

Take Amount100 µg -Amount300 µg of the sample to perform a two-step enzymatic digestion using Concentration25 millimolar (mM) Tris Ph8.5 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 Temperature37 °C for Duration02:00:00 .

2h
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 Temperature37 °C DurationOvernight .

14h
Digested samples can be stored at Temperature-80 °C .

Desalting the Peptide
Desalting the Peptide
8h
8h
Desalt the digest with Waters Oasis® HLB Cartridge.
Condition the column with Amount1 mL each of Concentration100 % (v/v) acetonitrile, Concentration80 % (v/v) acetonitrile with Concentration0.1 % (v/v) acetic acid, and Concentration0.1 % (v/v) acetic acid sequencially.
Adjust the sample to Concentration1 % (v/v) TFA, and load the sample onto the desalting column.
Wash the sample with Amount200 µL of Concentration0.1 % (v/v) acetic acid 3 times.
Elute peptides off the column with 150uL of Concentration40 % (v/v) acetonitrile with Concentration0.1 % (v/v) acetic acid, followed by 150uL of Concentration80 % (v/v) acetonitrile with Concentration0.1 % (v/v) acetic acid.
Put the sample in the SpeedVac until dry.
TMT Labeling
TMT Labeling
4h
4h
4h
Resuspend the sample in Concentration100 millimolar (mM) TEAB Ph8.5 and then add assigned TMT tag to the sample.

Incubate the sample with its associated TMT tag for Duration01:00:00 at room temperature.

Quench the reaction with Concentration5 % (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 Concentration200 millimolar (mM) ammonium formate and perform LC-MS analysis.