Jan 27, 2025

Public workspaceFFPE sections SP3 digestion -rapizyme and ACN

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldr8d8g5b/v1
FFPE sections SP3 digestion -rapizyme and ACN
  • Georgios Mermelekas1,
  • Mahshid Zarrineh1,
  • Rui Branca1,
  • Fabio Socciarelli2
  • 1Karolinska Institutet;
  • 2Weill Cornell Medical College
Mahshid Zarrineh
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldr8d8g5b/v1
Protocol Citation: Georgios Mermelekas, Mahshid Zarrineh, Rui Branca, Fabio Socciarelli 2025. FFPE sections SP3 digestion -rapizyme and ACN. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr8d8g5b/v1
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
Main workhorse protocol used in our proteomics lab for FFPE samples since 2023.
Created: December 12, 2024
Last Modified: January 27, 2025
Protocol Integer ID: 115111
Keywords: FFPE, SP3, proteomics, trypsin, rapizyme, magnetic beads, urea-free, peptides by trypsin, digestion of protein, protein, protein clean, peptide, peptide level, ffpe, acn this procedure, digestion, ultrasonication step, rapizyme from water, procedure, urea during digestion
Funders Acknowledgements:
Swedish Research Council
Grant ID: 2019-04830
Erling Perssons stiftelse
Grant ID: 22/9-2020
Cancerfonden
Grant ID: 23 2819 Pj 01 H
Abstract
This procedure takes as input Formalin-Fixed Paraffin-Embedded (FFPE) samples, typically 2 to 5 slices of 5μmx5mmx5mm. It begins with lysis/deparaffinization of the samples including an ultrasonication step, followed by protein clean-up with sp3-beads, denaturation, reduction and alkylation of cysteines, followed by digestion of proteins to peptides by trypsin (rapizyme from Waters), while using 10% ACN instead of urea during digestion, and ends with peptide level sp3 cleanup.
Materials
  • Reagents and solvents:

ReagentTrizma® baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1503
ReagentTRIS Buffer, 1.0M, pH 8.0, Molecular Biology GradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #648314-100ML

ReagentHydrochloric acid 1.000 lMerck Millipore (EMD Millipore)Catalog #109911

ReagentSDS 20% solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F
Sodium n-dodecyl sulfate (SDS), 20% aq. soln.

ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43815
DL-Dithiothreitol, threo-1,4-Dimercapto-2,3-butanediol
Product number: 43815-1G
Molecular Weight: 154.25 g/mol
  • Use the BioUltra grade powder to prepare 1M aqueous stock solution; aliquots can be prepared and frozen.

Reagent2-ChloroacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0267
2-Chloroacetamide
CAS No.: 79-07-2
Molecular Weight: 93.51 g/mol
  • Use crystalline powder (e.g., Sigma-Aldrich Cat. No. C0267, ≥98%) to prepare fresh aqueous solution immediately before use

ReagentCytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic, 65152105050250Fisher ScientificCatalog #11819912
(Sigma-Aldrich P/N: GE65152105050250)

ReagentCytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophilic, 45152105050250Fisher ScientificCatalog #11548692
(Sigma-Aldrich P/N: GE45152105050250)

ReagentGibco™ HEPES (1M)Fisher ScientificCatalog #15-630-080
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
CAS No.: 7365-45-9
Molecular Weight: 238.30 g/mol
  • Use commercially available 1M aqueous stock solution (e.g., Gibco, Fisher Scientific Cat No. 15-630-080; 100 mL)

ReagentCalcium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #21108-500G
ReagentWater, Optima™ LC/MS Grade, Fisher Chemical™Thermo Fisher ScientificCatalog #AAB-W6-4
  • Use ultra-pure water - we recommend Fisher Optima LCMS grade (Fisher Scientific P/N 10505904)

ReagentAcetonitrile, Optima LC/MS grade, Fisher ChemicalFisher ScientificCatalog #A955212
CAS No.: 75-05-8
  • Use LCMS-grade, e.g. Acetonitrile Optima LCMS grade (Fisher Scientific P/N 10055454) or HPLC grade S (Rathburn)

ReagentEthanol, 99.8%, for HPLC, absolute, Thermo Scientific ChemicalsFisher ScientificCatalog # 12347163

ReagentRapiZyme Trypsin, MS Grade, 4/pk (100ug)WatersCatalog #186010108


  • Plastics and equipment:
ReagentEppendorf Safe-Lock micro test tubes 2mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123344
ReagentEppendorf Safe-Lock micro test tubes 1.5mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123328
ReagentEppendorf® Protein LoBind Tubes, 1.5mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030108442-1EA

FiveEasy Plus pH meter, METTLER TOLEDO.

Ultrasonic processor (Twitter), UP200ST, Hielscher.

Vortex-Genie‱ 2 mixer, AC/DC input 220 V AC, Schuko plug, Merck, cat Number: Z258423-1EA.

VWR‱ MiniStar/MiniStar blueline, Spin-down/Microcentrifuge, VWR art.nr: 521-2161P, VWR.

Eppendorf‱ ThermoMixer‱ C, Product Number: EP5382000015-1EA, Merck.

ELMI Intelli-Mixer‱ , RM-2S, ELMI tech.

Centrifuge, 5430/ 5430 R, eppenorf.
Troubleshooting
Reagent preparation
1h 10m
Preparing 1M Tris-HCl pH8
Prepare Amount10 mL of Concentration1 Molar (M) aqueous solution of Tris-base.
  • Weigh approximately Amount1.211 g of Trizma‱ base powder put in 10 mL Volumetric flask.
  • Fill the flask up to the etched ring mark.
  • Mix thoroughly until dissolved.
  • Calibrate pH meter.
  • Adjust the pH of the solution to Ph8 using HCl solution.

Alternatively, use premade TRIS buffer Ph8.0 Concentration1 Molar (M) from Merck.


20m
Lysis Buffer preparation
Final buffer concentration: 4%SDS, Concentration0.2 Molar (M) Tris-HCL Ph8 , Concentration0.2 Molar (M) DTT
To have the above final concentration in 1 mL volume, use the following volumes and fill up to Amount1 mL with water:
Amount200 µL SDS 20%
Amount200 µL DTT 1M
Amount200 µL Tris-HCl 1M pH 8
5m
Alkylation reagent preparation
Prepare Amount5 mL of Concentration750 millimolar (mM) aqueous solution of chloroacetamide (CAA).
  • weigh approximately Amount350.6 mg of the microspatula of CAA powder.
  • add the exact volume of water to a final concentration of Concentration750 millimolar (mM)
  • mix thoroughly (vortex) until dissolved
  • pulse-spin in a microcentrifuge
Note
Example
dissolving in 5 ml of water gives the desired 750 mM concentration.

Note
The volumes of reagents can be adjusted to the number of samples.

5m
Preparing 1M CaCl2
Prepare Amount1 mL of Concentration1 Molar (M) aqueous solution of calcium chloride hexahydrate (CaCl2).
  • weigh approximately Amount219 mg of calcium chloride hexahydrate (CaCl2) powder.
  • add the exact volume of water to a final concentration of Concentration1 Molar (M)
  • mix thoroughly (vortex) until dissolved
  • pulse-spin in a microcentrifuge

Note
You can aliquot the solution and keep them in freezer.

5m
Digestion Buffer preparation
Final buffer concentration: Concentration50 millimolar (mM) HEPES Ph7.8 , Concentration10 millimolar (mM) CaCl2 , Concentration10 % (v/v) ACN )
To have the above final concentration in 1 mL volume, use the following volumes and fill up to Amount1 mL with water:
Amount50 µL HEPES 1M pH 7,8
Amount100 µL ACN
Amount10 µL 1M CaCl2
5m
SP3-bead slurry preparation
(sufficient for 10 samples)
15m
Take out the two bottles with Sera-Mag speed beads from the fridge and keep them at room temperature for 10 minutes. Duration00:10:00
10m
Shake the two SP3 bead bottles gently (without vortex, until all beads are suspended in liquid, takes about 5 minutes).Duration00:05:00
5m
Combine Amount50 µL of each bead type in a clean Safe-lock Eppendorf tube (1.5mL or 2mL).
Wash the beads:
  • Place the tube with the bead mixture on a magnetic rack and let the beads settle for 2 minutes.Duration00:02:00
  • Remove and discard the supernatant.
  • Rinse the beads with Amount500 µL water by gentle pipette mixing (off the magnetic rack).
  • Repeat wash steps two more times.
  • Re-suspend and store the beads in Amount500 µL water in Temperature4 °C
Note
The washed beads can be stored in +4ºC up to two weeks. Beads can handle low and high pH, and can be safely heated up to 60ºC.
!!! Never freeze the beads.
!!! Never sonicate the beads.

Deparaffinization - Protein extraction from FFPE tissue sections in tubes
1h 34m 30s
Add Amount200 µL of lysis buffer to each FFPE sample.

Note
This protocol is originally written for use with the Twitter Ultrasonic processor from Hielscher, UP200ST. If a Covaris AFA ultrasonicator is available that is also a good option.

Bear in mind that optimal sample capacities are different:
Covaris sonication will have a capacity of 100μl of lysate maximum. Hielscher sonication can take up to 1mL if necessary. However, 200μl is recommended as maximum due to volume constraints in subsequent steps of the protocol. (see steps 16 and 18 below).

As such, for Covaris, a good size of FFPE sample input is 2 slices of 5μmx5mmx5mm. (about 10μg to 20μg of protein).
For Hielscher, sample input of 5 such slices is recommended. (about 25μg to 50μg of protein).

5m
Be sure every sample is submerged in the lysis buffer, otherwise vortex or shake them.
Heat the samples in the heating block at Temperature95 °C for Duration00:30:00 (30min) in agitation, 400 rpm.
30m
Remove the samples from the heating block and let it cool down for some minutes at TemperatureRoom temperature .
2m
Spin down the samples and Sonicate the samples with Twitter sonicator with the following settings:
  • Set the Amplitude (A) to 100%
  • Set the pulsation mode (C) to 100%
  • Period Clock: On
  • On Time: 60s
  • Off Time: 30s
  • Limit Type: Time
  • Limit Value: To perform 10 cycles you need to set Duration00:15:00 (15min) .
Note
Calculating Limit Value:
(60s On Time + 30 Off Time) * Number of Cycles

For example:
For 10 Cycles: (60+30)*10= 900s = You should set 15 min on instrument.

  • IMPORTANT: Ensure all positions on the Twitter sonicator are occupied. If you do not have enough samples to fill the positions, use tubes filled with water to balance the instrument.


15m
Heat the samples in the heating block at Temperature95 °C for Duration00:15:00 (15min) in agitation, 400 rpm.
15m
Spin down the samples and Sonicate the samples with twitter with the following settings:
  • Set the Amplitude (A) to 100%
  • Set the pulsation mode (C) to 100%
  • Period Clock: On
  • On Time: 60s
  • Off Time: 30s
  • Limit Type: Time
  • Limit Value: To perform 5 cycles you need to set Duration00:07:30 .
Note
IMPORTANT: Ensure all positions on the Twitter sonicator are occupied. If you do not have enough samples to fill the positions, use tubes filled with water to balance the instrument.

7m 30s
Centrifuge at Centrifigation13000 x g, 23°C for Duration00:10:00 (10 min) at TemperatureRoom temperature .

10m
Recover the supernatant and put in a new Safe-lock 2mL Eppendorf tube.
10m
SP3 digestion of FFPE samples
17h 14m
Add Amount300 µL 750 mM chloroacetamide to each sample, vortex, and incubate for Duration00:10:00 (10min) at TemperatureRoom temperature , keeping the samples in the fume hood.
Note
The volume of 300 μl here is expecting the recommended lysate volume of 200μl (see step 7 above). If the lysate volume is different, the needed volume of chloroacetamide solution to be added should be adjusted accordingly.
μμ
15m
Add Amount50 µL of beads suspension to each sample and pipette gently to resuspend them.
10m
Add Amount1500 µL ACN to the samples  (acetonitrile concentration of 73%).
Note
Concerning volumes, given that the maximum volume capacity of the Eppendorf tube is 2mL, consider the volumes used in steps 7 and 16.

2m
Incubate the suspension in a rotating rack for Duration00:20:00 (20 min) at TemperatureRoom temperature .
20m
At the end of the incubation remove the tubes from the rack, shake them to detach the beads from the lid and incubate in a magnetic rack for Duration00:02:00 (2 min).
5m
Aspirate and discard the supernatant, without removing the tube from the rack.
1m
While maintaining the tubes on the magnetic rack, flush the beads with Amount500 µL Ethanol 70% and incubate Duration00:00:30 (30s), then aspirate the liquid phase and discard it.

2m
Repeat the previous step.
3m
While maintaining the tubes on the magnetic rack, flush the beads with Amount500 µL ACN , incubate Duration00:00:30 (30s), aspirate and discard the supernatant.
3m
Let the beads dry for Duration00:00:20 (20s), then close the lid to avoid excessive drying.
1m
Dissolve RAPIZYME powder (Amount100 µg of trypsin) in Amount100 µL of Fisher Optima water (quick vortex or pipette up and down). Add the Amount100 µL of trypsin solution to Amount1900 µL of digestion buffer.
Note
If the entire 100 μg of trypsin are to be used up in one experiment, then one can directly dissolve in the digestion buffer.
Otherwise, leftover Rapizyme dissolved in water can be kept in the -20C freezer and be used later (up to a month). Beware of activity degradation due to freeze/thaw cycles, so ideally plan ahead and freeze in aliquots to be used up in single occasions.


2m
Add Amount100 µL from the trypsin containing digestion buffer to each sample (i.e. onto the dry beads).
Incubate for Duration16:00:00 (16h) at Temperature37 °C with mild shaking, using an oven type incubator (i.e. avoid temperature gradient between the bottom and the top of the tube).

16h
SP3 peptide clean-up, peptide concentration
52m
After overnight incubation, add Amount2 mL acetonitrile to reach a concentration of more than Concentration95 % (v/v) ACN.

Note
In order guarantee the binding of all peptides to the SP3 beads, the organic content composition of the liquid must be >95%.
2m
Incubate the tubes in a gently rotating rack for 20min Duration00:20:00 at TemperatureRoom temperature .
20m
After incubation, shake the tubes to recover the beads stuck on the lid, then incubate the tubes on a magnetic rack for Duration00:02:00 (2 min).
5m
While maintaining the tubes on the magnetic rack, aspirate and discard the liquid phase, then flush the beads with Amount500 µL acetonitrile and wait for Duration00:00:30 (30s).
2m
Repeat the previous step.
3m
While maintaining the tubes on the magnetic rack, aspirate and discard the liquid phase, then let it dry a few seconds.

10m
Take the tubes off the magnetic rack, and resuspend inAmount50 µL of MS grade water. Mix gently by brief vortex or pipetting and spin down.
Incubate the tube on the magnetic rack for Duration00:02:00 (2 min), then recover the liquid phase and put it in a new Low-bind 1.5mL Eppendorf tube.
5m
Repeat the previous step, to be sure of removing all the beads.
5m
Perform the peptide concentration measurement, using Optima LCMS grade water as blank.
Protocol references
1. Hughes, C.S., et al., Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol, 2014. 10(10): p. 757.
2. Hughes, C.S., et al., Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc, 2019. 14(1): p. 68-85.
3. Coscia, F., et al., A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis. J Pathol, 2020. 251(1): p. 100-112.
4. Stanojevic, A., et al., Data-Independent Acquisition Mass Spectrometry Analysis of FFPE Rectal Cancer Samples Offers In-Depth Proteomics Characterization of the Response to Neoadjuvant Chemoradiotherapy. Int J Mol Sci, 2023. 24(20): p. 15412