Nov 22, 2024

Public workspaceSP3 protein cleanup - digestion w/ rapizyme and ACN - SP3 peptide cleanup

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.j8nlk9en1v5r/v1
  • 1Karolinska Institutet;
  • 2Science for Life Laboratory
Rui Branca
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk9en1v5r/v1
Protocol Citation: Georgios Mermelekas, Konstantin Barylyuk, Eduardo Araújo, Rui Branca 2024. SP3 protein cleanup - digestion w/ rapizyme and ACN - SP3 peptide cleanup. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9en1v5r/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 since 2023.
Created: November 13, 2024
Last Modified: November 22, 2024
Protocol Integer ID: 112049
Keywords: proteomics, sample preparation, digestion, cleanup, protein extraction, reduction, alkylation, SP3
Abstract
This procedure takes as input samples (cells, tissue, tumor piece, biopsy, etc) previously lysed in SDS-based lysis buffer (4% (w/v) SDS, 25 mM HEPES pH 7.6, 1mM DTT) and with DNA already sheared, and it delivers tryptic peptides in water. It consists of 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:

ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 or
ReagentDTT (DL-Dithiothreitol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #43819
DL-Dithiothreitol, threo-1,4-Dimercapto-2,3-butanediol
CAS No.: 3483-12-3
Molecular Weight: 154.25 g/mol
  • Use powder, molecular biology grade or similar (e.g., Sigma-Aldrich Cat. No. 43819, ≥99% (RT)) to prepare 1M aqueous stock solution; aliquots can be prepared and frozen

Reagent2-ChloroacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0267
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

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 powder, molecular biology grade or similar (≥98.5%) to prepare 1M aqueous stock solution (pH 7.8)
or
  • Use commercially available 1M aqueous stock solution (e.g., Gibco, Fisher Scientific Cat No. 15-630-080; 100 mL)


ReagentWater, Optima LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #W6212
  • 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)

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)

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

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

ReagentSodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2OMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F


  • Plastics and equipment:

ReagentEppendorf Safe-Lock micro test tubes 1.5mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123328
ReagentEppendorf Safe-Lock micro test tubes 2mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123344
Vortex-Genie‱ 2 mixer, AC/DC input 220 V AC, Schuko plug, Merck, cat Number: Z258423-1EA.

Protocol materials
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375-500G
Step 16
ReagentWater Optima™ LC/MS Grade Fisher Chemical™Fisher ScientificCatalog #W6-4
Before starting
ReagentEthanol, 99.8%, for HPLC, absolute, Thermo Scientific ChemicalsFisher ScientificCatalog # 12347163
Materials
ReagentEppendorf Safe-Lock micro test tubes 2mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123344
Materials
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Materials
ReagentCytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic, 65152105050250Fisher ScientificCatalog #11819912
Materials
ReagentRapiZyme Trypsin, MS Grade, 4/pk (100ug)WatersCatalog #186010108
Materials
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375-500G
Step 5
ReagentAcetonitrile, Optima LC/MS grade, Fisher ChemicalFisher ScientificCatalog #A955212
Materials
ReagentSDS 20% solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F
Step 5
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43815
Step 5
ReagentGibco™ HEPES (1M)Fisher ScientificCatalog #15-630-080
Materials
ReagentRapiZyme Trypsin, MS Grade, 4/pk (100ug)WatersCatalog #186010108
Step 16
ReagentCaCl2 hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #21108-500G
Step 16
ReagentSodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2OMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F
Materials
ReagentEppendorf Safe-Lock micro test tubes 1.5mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030123328
Materials
ReagentWater, Optima LC/MS Grade, Fisher ChemicalFisher ScientificCatalog #W6212
Materials
Reagent2-ChloroacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0267
Materials
ReagentDTT (DL-Dithiothreitol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #43819
Materials
ReagentCytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophilic, 45152105050250Fisher ScientificCatalog #11548692
Materials
Safety warnings
SP3 Beads can handle low and high pH, and can be safely heated up to 60ºC. However:
!!! Never freeze the beads.
!!! Never sonicate the beads.
Before start
This protocol assumes the cell lysis has been done and as such lysis buffer is already available. If not, prepare lysis buffer (4% (w/v) SDS, 25 mM HEPES 7.8±0.2, 1mM DTT) before starting.
All water used throughout is ReagentWater Optima™ LC/MS Grade Fisher Chemical™Fisher ScientificCatalog #W6-4

Reagent preparation
Reagent preparation
36m
36m
Reducing reagent preparation
Freshly prepare at leastAmount100 µL of Concentration100 millimolar (mM) aqueous solution of dithiothreitol (DTT).
  • weigh approximately Amount1-2 mg or one spoonful of a microspatula of DTT powder.
  • add the exact volume of water so as to reach a final concentration of Concentration100 millimolar (mM) .
  • mix thoroughly (vortex) until dissolved.
  • pulse-spin in a microcentrifuge.
Note
Example:
dissolving in 100 µl of water gives the desired 100 mM concentration.

Note
This may not be needed if lysis buffer already contains 1 mM DTT.


5m
Optional
Alkylation reagent preparation
Prepare Amount400 µL of Concentration200 millimolar (mM) aqueous solution of chloroacetamide (CAA).
  • weigh approximately Amount7-8 mg or four spoonfuls of the microspatula of CAA powder
  • add the exact volume of water to a final concentration of Concentration200 millimolar (mM)
  • mix thoroughly (vortex) until dissolved
  • pulse-spin in a microcentrifuge
Note
Example
dissolving in 400 µl of water gives the desired 200 mM concentration.

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







5m
SP3-bead slurry preparation
(sufficient for 20 samples)
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).
1m
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.


10m
Protein denaturation, reduction and alkylation of thiol groups
Protein denaturation, reduction and alkylation of thiol groups
35m
35m
Take an aliquot of protein extract (sample lysate) containingAmount200 µg of proteins and transfer to a clean tube (Eppendorf safelock 2mL). Takes about 10minDuration00:10:00 to do 20 samples.
Note
Make sure to use a 2mL Eppendorf tube, because the 2mL capacity will be needed later.


10m
Dilute the samples with lysis buffer (Concentration4 % (m/v) SDS, Concentration50 millimolar (mM) HEPES Ph7.8 7.8±0.2, Concentration1 millimolar (mM) DTT)ReagentSDS 20% solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375-500G ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43815
to get the same volume in all samples (Amount200 µL ). Final protein concentration is Concentration1 µg/µL .

10m
Add Amount10 µL of Concentration200 millimolar (mM) chloroacetamide, so as to reach a final concentration of Concentration10 millimolar (mM) chloroacetamide.

5m
Vortex to mix, then spin down briefly. Incubate for 10 min.Duration00:10:00

15m
Protein level SP3 cleanup
Protein level SP3 cleanup
45m
45m
Add SP3 bead solution to the protein sample using a ratio of 1:10 of bead solution : protein solution.
E. g. add Amount20 µL of SP3 beads solution to Amount200 µL of protein solution (containing Amount200 µg protein). Mix gently by pipetting. Takes about 10min Duration00:10:00 to do 20 samples.

10m
Add acetonitrile (ACN) to obtain a final ACN composition of ≥Concentration70 % (v/v) (e. g. Amount700 µL ).

2m
Incubate for 20minDuration00:20:00 in the continuously (and gentle) rotating rack.

20m
Place the tubes in the magnetic rack and incubate for 2 minutes Duration00:02:00 .

2m
Remove and discard the supernatant.
2m
Add Amount200 µL of Concentration70 % (v/v) EtOH and incubate for 30s Duration00:00:30 in magnetic rack. Remove and discard supernatant.

3m
Repeat previous step.
3m
Add 200 μl of neat ACN and incubate for 15s Duration00:00:15 in magnetic rack. Remove and discard supernatant, and air dry the beads for 30s Duration00:00:30 . Make sure you close the lid to avoid over-drying of the beads.

3m
Protein digestion
Protein digestion
17h 7m
17h 7m
Reconstitute the beads inAmount100 µL of digestion solution (Concentration50 millimolar (mM) HEPES Ph7.8 pH7.8±0.2, Concentration10 millimolar (mM) CaCl2, Concentration10 % (v/v) ACN, Amount4 µg Rapizyme (Waters)). Mix gently by pipetting.
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375-500G
ReagentCaCl2 hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #21108-500G
ReagentRapiZyme Trypsin, MS Grade, 4/pk (100ug)WatersCatalog #186010108

10m
Incubate for 16h Duration16:00:00 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
Day 2 - peptide level SP3 cleanup
Day 2 - peptide level SP3 cleanup
1h 24m 15s
1h 24m 15s
Take the tubes from the incubator, and allow to cool down to room temperature. Spin down briefly.
Add Amount1900 µL of ACN to the samples to reach final content of > Concentration95 % (v/v) .
Note
In order guarantee the binding of all peptides to the SP3 beads, the organic content composition of the liquid must be >95%.





Incubate for 20min Duration00:20:00 in the continuously (and gentle) rotating rack.
20m
Place the tubes in the magnetic rack and incubate for 2 minutes Duration00:02:00 .

2m
Remove and discard the supernatant.
Add 200 μl of neat ACN and incubate for 15s Duration00:00:15 in magnetic rack. Remove and discard supernatant.
15s
Repeat previous step.
Add Amount200 µL of water and mix gently by brief vortex and spin down.

Place on magnetic rack for 2 minDuration00:02:00 . Transfer supernatant to new tubes (1.5mL). 

Note
If sample is to proceed directly to LCMS vial, repeat this step. The point is to avoid any remaining residual magnetic beads to be carried over into the LC system.

2m
Determine peptide concentration with Bio-Rad Dcc. The BSA standard should be prepared in water.
1h
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.