Mar 14, 2022
SP3 protocol for proteomic analysis of tendon cryosections
This protocol is a draft, published without a DOI.
- Stefania Wunderli1,
- Jess Snedeker2,
- The Tendon Seed Network2
- 1ETHZ - ETH Zurich;
- 2University of Zurich
Protocol Citation: Stefania Wunderli, Jess Snedeker, The Tendon Seed Network 2022. SP3 protocol for proteomic analysis of tendon cryosections. protocols.io https://protocols.io/view/sp3-protocol-for-proteomic-analysis-of-tendon-cryo-bzndp5a6
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: November 01, 2021
Last Modified: March 14, 2022
Protocol Integer ID: 54693
Keywords: Proteomics, Cryosection, Tissue Lysis
Funders Acknowledgement:
Chan Zuckerberg Initiative
Grant ID: CZI2019-002426
Abstract
This protocol details the preparation of cryosection samples and laser capture microdissection (LCM) samples collected with a MMI Laser-Microdissection device for MS/MS analysis. The sample preparation includes tissue lysis, Sp3 technology (single-pot, solid-phase-enhanced sample preparation), trypsin digestion, C18-clean-up and peptide resuspension.
Attachments
ft3rbkj7p.docx
20KB
Materials
Reagents:
- Needle (1.2x40mm)
- RIPA buffer (Sigma)
- 100 millimolar (mM) TCEP
- 500 millimolar (mM) IAA
- MS water
- 80 % EtOH
- 100 % EtOH
- Carboxylated Magnetic Beads (hydrophilic and hydrophobic)
- SpeedBead Magnetic Carboxylated, modified particles, Ref: 65152105050250 (15 mL , azide 0.05%, Sigma (?))
- SpeedBead Magnetic Carboxylated, modified particles, Ref: 45152105050250 (15 mL , azide 0.05%, Sigma (?))
- Ammonium bicarbonate (ABC) 500 millimolar (mM) 100 µL of 500 millimolar (mM) ABC + 900 µL MilliQ
- Trypsin (0.1 µg/µL in 50 millimolar (mM) ABC) -> dissolved in 200 µL
- 5% TFA
- MS buffer (3% ACN, 0.1% formic acid)
- iRT peptides (Biognosys) (4 °C )
- Waters glass vial
Preparing cryosection samples
Preparing cryosection samples
1h 11m
1h 11m
Fill a 200 µL PCR tube with 100 µL 100% EtOH.
Wetten the dry tissue section on the glass slide with ca. 50 µL 100% EtOH.
Use a 1.2x40mm needle (18Gx1.5) to gently scrape off the tissue section
Transfer the scraped off tissue to EtOH in PCR tube.
Spin down 00:01:00 at 12000 x g .
1m
Remove 100% EtOH supernatant from tubes by using a 200 µL pipette.
Heat the tubes on thermocycler at 50 °C for ca.00:10:00 .
10m
Continue with A) or B).
A) Tissue lysis LCM samples51 steps
Add 20 µL RIPA lysis buffer to each sample: Add to tube, close tubes and shake down the liquid onto cap.
Preparing cryosection samples
Preparing cryosection samples
1h 21m 30s
1h 21m 30s
Incubate samples at 95 °C on thermocycler for 01:00:00 .
1h
LCM samples: upside-down, using magnetic rack for SP3 as a holder and stick tubes to rack with a tape.
Centrifuge LCM samples for 00:10:00 at 2000 x g .
10m
Take 5 µL of RIPA in each sample tube, pipet onto cap and scrape off remaining tissue from cap. Pipet RIPA back into tube. (LCM samples only).
Spin down for 00:00:30 at 2000 x g .
30s
Check under microscope, whether tissue pieces are still sticking to the cap.
Pool all the blank samples into one tube (ca.40 µL in total).
Reduction and alkylation
Reduction and alkylation
30m
30m
Note
Final volume/concentrations:
- -2 millimolar (mM) TCEP: Add 0.4 µL of 100 millimolar (mM) TCEP solution to
20 µL of sample.
- 15.5 millimolar (mM) IAA: Add 0.622 µL of 500 millimolar (mM) IAA solution to 20 µL of sample.
Prepare a reduction/alkylation stock solution for TCEP and IAA with the final concentrations from above.
8.8 µL TCEP + 13.684 µL IAA
Add 1.022 µL of reduction/alkylation stock solution to each sample (if volume is 20 µL ),
add 2.044 µL to blank.
Vortex
Incubate samples at 60 °C for 00:30:00 at 600 rpm .
30m
Spin down samples quickly.
Sp3 assisted protein capture and clean-up
Sp3 assisted protein capture and clean-up
45m
45m
Note
Hydrophilic and hydrophobic beads come at a stock concentration of 50 µg/µL . Use 1 µL of stock solution (10 µg/µL ) for each µg of protein.
LCM samples: assuming a protein amount of 10 µg per sample we need 10 µL stock solution per sample: 15(+1) samples x 10 µL = 160 µL stock.
Cryosection samples: ca. 60 µL total protein per sample, so we need 60 µL stock solution: 4 samples (+1) x 40 µL = 200 µL .
Prepare: 360 µL at 10 µg/µL = 3600 µg beads in total (1800 µg hydrophilic beads + 1800 µg hydrophobic beads).
Prepare the Carboxylated Magnetic Beads stock solution (360 µL ) with a concentration of 10 µg/µL .
Gently shake flasks with beads to resuspend bottom layer.
Mix hydrophilic and hydrophobic beads at a 1:1 ratio: 36 µL hydrophilic and 36 µL hydrophobic beads (conc: 50 µg/µL ).
Put beads on rack and remove liquid.
Wash the beads 3 times with water at a concentration of 50 µg/µL : 3600 µg beads
at 5 µg/µL = 720 µL total volume.
Resuspend the beads in water at a working concentration of 10 µg/µL : 3600 µg beads
at 10 µg/µL = 360 µL water total.
Protein binding to beads.
Add 100% EtOH to the sample to reach 80% EtOH (v/v).
- LCM samples: add 80 µL
- Cryosection samples: add 400 µL
Add magnetic beads stock to samples (stock concentration: 10 µg/µL , 100 µg or600 µg beads)
- LCM samples: add 10 µL
- Cryosection samples: 60 µL
Incubate for 00:45:00 at Room temperature and 800 rpm .
45m
Spin down quickly.
Wash beads.
Insert the tubes into magnetic stand to collect the magnetic beads.
Remove supernatant and discard.
Add 80% EtOH (2 times the sample volume):
- LCM samples: use 200 µL
- Cryosection samples: use 960 µL
Shake 00:03:00 at Room temperature , 800 rpm .
3m
Collect beads in-between washed on magnetic stand, discard wash solution.
Repeat in total 3x.
Trypsin Digestion
Trypsin Digestion
- Make a trypsin stock solution in 50 millimolar (mM) ABC for LCM and cryosection samples separately:
- LCM samples: final concentration = 0.005 µg/µL , final volume = 15(+1) * 20 µL = 320 µL
Mix 304 µL ABC (50 millimolar (mM) ) with 16 µL trypsin (at 0.1 µg/µL in 500 millimolar (mM) ABC).
- Cryosection samples: final concentration = 0.025 µg/µL , final volume = 4 * 60 µL (+10 µL ) = 250 µL
Mix 187.5 µL ABC (50 millimolar (mM) ) with 62.5 µL trypsin (at 0.1 µg/µL in 500 millimolar (mM) ABC).
Add trypsin stock solution to each sample:
- LCM samples: add 20 µL to LCM samples (contains ca.0.1 µg trypsin).
- Cryosection samples: add 60 µL l to reference samples (contains 1.2 µg trypsin).
Digest overnight at 37 °C .
Peptide Extraction
Peptide Extraction
15m
15m
Spin the tubes down quickly.
Place tubes onto magnetic stage and transfer supernatant into a new 1.5 mL Eppendorf tube.
Add ddH2O to magnetic beads
- LCM samples: add 30 µL
- Cryosection samples: add 70 µL
Sonicate for 00:15:00 .
15m
Spin down quickly.
Combine supernatant in the same Eppendorf tube as in step 1 above.
Quench digestion by adding 5% TFA
- LCM samples: add 10 µL
- Cryosection samples: add 26 µL
Check pH.
Drying and resuspension
Drying and resuspension
10m
10m
Snap freeze samples and place open collection tube in a vacuum evaporator (until completely dry).
Freeze samples or resuspend.
For resuspension: Prepare iRTs in MS buffer at a ratio of 1:20.
Resuspend samples in 10 µL (LCM) or 20 µL (cryo) MS buffer + iRT.
Sonicate for 00:10:00 at Room temperature .
10m
Spin down quickly.
Transfer the whole sample into a Waters glass vial.