TITLE: SP3 protocol for proteomic analysis of tendon cryosections
AUTHORS: Stefania  Wunderli, Jess  Snedeker, The Tendon Seed Network

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparing cryosection samples
1. Fill a 200 µL PCR tube with 100 µL 100% EtOH.

SECTION: Preparing cryosection samples
2. Wetten the dry tissue section on the glass slide with ca. 50 µL 100% EtOH.

SECTION: Preparing cryosection samples
3. Use a 1.2x40mm needle (18Gx1.5) to gently scrape off the tissue section

SECTION: Preparing cryosection samples
4. Transfer the scraped off tissue to EtOH in PCR  tube.
SECTION: Preparing cryosection samples
5. Spin down 1 minat 12000 x g.
SECTION: Preparing cryosection samples
6. Remove 100% EtOH supernatant from tubes by using a 200 µL pipette.

SECTION: Preparing cryosection samples
7. Heat the tubes on thermocycler at 50 °C for ca.10 min.

SECTION: Preparing cryosection samples
8. Continue with A) or B).
SECTION: Preparing reference samples
9. Add 20 µL RIPA lysis buffer to each sample.

SECTION: Preparing cryosection samples
9. Add 20 µL RIPA lysis buffer to each sample: Add to tube, close tubes and shake down the liquid onto cap.

SECTION: Preparing cryosection samples
11. Incubate samples at 95 °C on thermocycler for 60 min.

SECTION: Preparing cryosection samples
11.1. LCM samples: upside-down, using magnetic rack for SP3 as a holder and stick tubes to rack with a tape.
9.1. 
SECTION: Preparing cryosection samples
12. Centrifuge LCM samples for 10 min at 2000 x g.

SECTION: Preparing cryosection samples
13. 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).

SECTION: Preparing cryosection samples
14. Spin down for 30 s at 2000 x g.

SECTION: Preparing cryosection samples
15. Check under microscope, whether tissue pieces are still sticking to the cap.
SECTION: Preparing cryosection samples
16. Pool all the blank samples into one tube (ca.40 µL in total).

SECTION: Preparing reference samples
10. Incubate samples at 95 °C on thermocycler for 60 min (cover tubes with metal plate to avoid condensation).

Spin down 1-2 times in between.

SECTION: Preparing reference samples
11. Prepare solutions for measurement of protein concentration by Qubit.

10 µL Qubit reagent
1990 µLQubit Buffer

SECTION: Preparing reference samples
12. Measure protein concentration according to FGCZ protocol.
SECTION: Reduction and alkylation
17. 
 

Prepare a reduction/alkylation stock solution for TCEP and IAA with the final concentrations from above.

8.8 µL TCEP + 13.684 µL  IAA
SECTION: Reduction and alkylation
18. Add 1.022 µL of reduction/alkylation stock solution to each sample (if volume is 20 µL), 
add 2.044 µL to blank.
SECTION: Reduction and alkylation
19. Vortex
SECTION: Reduction and alkylation
20. Incubate samples at 60 °C for 30 min at 600 rpm.
SECTION: Reduction and alkylation
21. Spin down samples quickly.
SECTION: Sp3 assisted protein capture and clean-up
22. 
 

Prepare the Carboxylated Magnetic Beads stock solution (360 µL) with a concentration of 10 µg/µL.
SECTION: Sp3 assisted protein capture and clean-up
22.1. Gently shake flasks with beads to resuspend bottom layer.
SECTION: Sp3 assisted protein capture and clean-up
22.2. Mix hydrophilic and hydrophobic beads at a 1:1 ratio: 36 µL hydrophilic and 36 µL hydrophobic beads (conc: 50 µg/µL).
SECTION: Sp3 assisted protein capture and clean-up
22.3. Put beads on rack and remove liquid.
SECTION: Sp3 assisted protein capture and clean-up
22.4. 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.
SECTION: Sp3 assisted protein capture and clean-up
22.5. Resuspend the beads in water at a working concentration of 10 µg/µL : 3600 µg beads 
at 10 µg/µL = 360 µL water total.
SECTION: Sp3 assisted protein capture and clean-up
23. Protein binding to beads.
SECTION: Sp3 assisted protein capture and clean-up
23.1. Add 100% EtOH to the sample to reach 80% EtOH (v/v).

LCM samples: add 80 µL
Cryosection samples: add 400 µL
SECTION: Sp3 assisted protein capture and clean-up
23.2. 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
SECTION: Sp3 assisted protein capture and clean-up
23.3. Incubate for 45 min at Room temperature and 800 rpm.

SECTION: Sp3 assisted protein capture and clean-up
23.4. Spin down quickly.
SECTION: Sp3 assisted protein capture and clean-up
24. Wash beads.
SECTION: Sp3 assisted protein capture and clean-up
24.1. Insert the tubes into magnetic stand to collect the magnetic beads.
SECTION: Sp3 assisted protein capture and clean-up
24.2. Remove supernatant and discard.
SECTION: Sp3 assisted protein capture and clean-up
24.3. Add 80% EtOH (2 times the sample volume):

LCM samples: use 200 µL
Cryosection samples: use 960 µL
SECTION: Sp3 assisted protein capture and clean-up
24.4. Shake 3 min at Room temperature, 800 rpm.

SECTION: Sp3 assisted protein capture and clean-up
24.5. Collect beads in-between washed on magnetic stand, discard wash solution.
SECTION: Sp3 assisted protein capture and clean-up
24.6. Repeat in total 3x.
SECTION: Trypsin Digestion
25. 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 µLABC (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 µLABC (50 millimolar (mM)) with 62.5 µL trypsin (at 0.1 µg/µL in 500 millimolar (mM) ABC).
SECTION: Trypsin Digestion
26. 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 µLl to reference samples (contains 1.2 µg trypsin).
SECTION: Trypsin Digestion
27. Digest overnight at 37 °C.
SECTION: Peptide Extraction
28. Spin the tubes down quickly.
SECTION: Peptide Extraction
29. Place tubes onto magnetic stage and transfer supernatant  into a new 1.5 mL  Eppendorf tube.
SECTION: Peptide Extraction
30.        Add ddH2O to magnetic beads

LCM samples: add 30 µL

Cryosection samples: add 70 µL

SECTION: Peptide Extraction
31. Sonicate for 15 min.
SECTION: Peptide Extraction
32. Spin down quickly.
SECTION: Peptide Extraction
33. Combine supernatant in the same Eppendorf tube as in step 1 above.
SECTION: Peptide Extraction
34.       Quench digestion by adding 5% TFA

LCM samples: add 10 µL

Cryosection samples: add 26 µL
SECTION: Peptide Extraction
35. Check pH.
SECTION: Drying and resuspension
36. Snap freeze samples and place open collection tube in a vacuum evaporator (until completely dry).
SECTION: Drying and resuspension
37. Freeze samples or resuspend.
SECTION: Drying and resuspension
38. For resuspension: Prepare iRTs in MS buffer at a ratio of 1:20.
SECTION: Drying and resuspension
39. Resuspend samples in 10 µL (LCM)  or 20 µL (cryo) MS buffer + iRT.

SECTION: Drying and resuspension
40. Sonicate for 10 min at Room temperature.

SECTION: Drying and resuspension
41. Spin down quickly.
SECTION: Drying and resuspension
42. Transfer the whole sample into a Waters glass vial.
10.