Protocol for label-free ribosome-associated protein enrichment from mammalian cells by RAPIDASH

Summary

Ribosome-associated proteins (RAPs) enable modulation of gene expression at the level of mRNA translation in response to cellular needs. Here, we describe a method called ribosome-associated protein identification by affinity to sulfhydryl-charged resin (RAPIDASH) for tag-free isolation of RAP-bound ribosomes from mammalian samples for mass spectrometry-based proteomics. Samples are first lysed and then undergo sucrose cushion ultracentrifugation and subsequent chromatography using a sulfhydryl-charged resin. While RAPIDASH is optimized for mammalian samples, we expect that it can be adapted for non-mammalian samples.

For complete details on the use and execution of this protocol, please refer to Susanto et al.¹

Graphical abstract

Highlights

• •Tag-free technique to enrich ribosomes and associated proteins in 5 h
• •Sucrose cushion ultracentrifugation procedure to pellet dense protein complexes
• •Steps for chromatography with sulfhydryl-charged resin to enrich ribosomes
• •Guidance on how steps may be adapted for other samples

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Ribosome-associated proteins (RAPs) enable modulation of gene expression at the level of mRNA translation in response to cellular needs. Here, we describe a method called ribosome-associated protein identification by affinity to sulfhydryl-charged resin (RAPIDASH) for tag-free isolation of RAP-bound ribosomes from mammalian samples for mass spectrometry-based proteomics. Samples are first lysed and then undergo sucrose cushion ultracentrifugation and subsequent chromatography using a sulfhydryl-charged resin. While RAPIDASH is optimized for mammalian samples, we expect that it can be adapted for non-mammalian samples.

Before you begin

The ribosome, the molecular complex that translates the genome into effector proteins, was historically viewed as a passive machine composed solely of approximately 80 core ribosomal proteins in eukaryotes and four ribosomal RNAs (rRNAs). This compositional definition was based in part on analysis of ribosomes isolated under high salt conditions that stripped away associating proteins.² Recent work, however, has discovered ribosome-associated proteins (RAPs) impart translational specificity and thus allow the ribosome to directly regulate gene expression.^3,4,5,6,7,8 These hundreds of RAPs link mRNA translation to many cellular processes, including regulation of translation fidelity,⁹ cell differentiation,^3,4,5,8 metabolism,^6,7 and the cell cycle.^6,10,11 However, the full complement of RAPs, their dynamics across sample types or upon stimuli, and their functions are poorly understood.

Innovation

Recent efforts to apply mass spectrometry-based proteomics to characterize RAPs across biological samples have resulted in methods that fall into two broad categories. The first consists of methods that separate cellular contents by size¹² or density.^13,14,15 These methods are simple to implement and can be used on any sample but are highly nonspecific due to the co-fractionation of non-ribosomal complexes. The second category consists of methods based on affinity purification via genetic⁶ or chemical⁸ handles that have been introduced to ribosomes. While these methods are exceptionally specific for ribosomes, genetic tagging of ribosomal proteins⁶ is in some cases difficult or impossible, and current approaches that rely on the introduction of chemical handles bias the enrichment for complexes at the early stages of translation.⁸

We have developed a technique called Ribosome-Associated Protein IDentification by Affinity to SulfHydryl-charged resin (RAPIDASH) to enrich RAP-bound ribosomes from any sample.¹ This label-free technique is much more specific than traditional fractionation methods. First, sample lysate undergoes sucrose cushion ultracentrifugation to pellet high density protein complexes, which are then subjected to chromatography with sulfhydryl-charged resin to enrich RNA-containing protein complexes, which are mostly ribosomes. Previously, the sulfhydryl-charged resin was used with vastly different conditions to purify ribosomes where the RAPs were stripped off.^1,16 We optimized the procedure to preserve the interactions between the ribosome and salt-sensitive RAPs by changing the buffers, the order of the steps, and the time and speed of centrifugation.

Technique application

The sulfhydryl-charged resin is generated by coupling cysteine to SulfoLink Coupling Resin (Thermo Fisher Scientific) in an alkaline buffer. The resulting thioether linkage is irreversible and resistant to reducing conditions (Figure 1). While the mechanism for RNA binding to the sulfhydryl-charged resin is not fully understood, poly(A)-enriched RNA does not bind to the sulfhydryl-charged resin,¹ and ribosome binding to the resin does not depend on mRNA or nascent polypeptide chains.¹ This technique has been applied to mouse embryonic stem cells (mESCs), mouse embryonic tissues (limb, liver, and forebrain), murine bone marrow-derived macrophages, and human cancer cells (PC3 cells) to identify how RAPs change across samples and upon acute stimuli.¹ The protocol below is specifically written for mESCs and is applicable to many cell types. Once the lysate is generated, the RAP enrichment can be completed within one day.

Figure 1.

Coupling reaction to generate the sulfhydryl-charged resin

SulfoLink Coupling Resin (Thermo Fisher Scientific) consists of agarose beads that display iodoacetyl groups at the end of a 12-atom spacer arm. When the resin is rocked with L-cysteine in an alkaline buffer, the thiolate of L-cysteine reacts with the iodoacetyl group to form a thioether linkage.

The values in the example protocol below are written for processing one 15-cm plate of E14 mESCs harvested at log-phase.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
2-mercaptoethanol	Gibco	Cat#21985023
Ammonium bicarbonate	Sigma-Aldrich	Cat#09830-500G
Ammonium chloride	Sigma-Aldrich	Cat#A9434-500G
Buffer kit, RNase-free	Invitrogen	Cat#AM9010
Cycloheximide	Sigma-Aldrich	Cat#C7698-1G
DPBS, no calcium, no magnesium	Gibco	Cat#14190250
EDTA (0.5 M), pH 8.0, RNase-free	Invitrogen	Cat#AM9262
EmbryoMax ES cell qualified FBS	Millipore	Cat#ES-009-B
EmbryoMax L-glutamine solution (100×), 200 mM	Millipore	Cat#TMS-002-C
EmbryoMax MEM, non-essential amino acids (100×)	Millipore	Cat#TMS-001-C
EmbryoMax penicillin-streptomycin solution, 100×	Millipore	Cat#TMS-AB2-C
Ethanol	Gold Shield Distributors	Cat#412804
Glycerol	Sigma-Aldrich	Cat#G9012-100ML
Halt protease and phosphatase inhibitor single-use cocktail, EDTA-free (100×)	Thermo Scientific	Cat#78443
HEPES	Sigma-Aldrich	Cat#H3375-100G
Hydrochloric acid	Fisher Scientific	Cat#A144SI-212
KnockOut DMEM	Gibco	Cat#10829-018
Laemmli-SDS sample buffer 6×, reducing	Bioworld	Cat#105700211
L-cysteine	Sigma-Aldrich	Cat#168149-25G
Magnesium acetate tetrahydrate	Sigma-Aldrich	Cat#M5661-250G
Mouse leukemia inhibitory factor (LIF) protein	GeminiBio	Cat#400-495
Pierce DTT (dithiothreitol), No-Weigh format	Thermo Scientific	Cat#A39255
Potassium hydroxide	Sigma-Aldrich	Cat#221473-500G
ProteoExtract protein precipitation kit	Sigma-Aldrich	Cat#539180-1KIT
RNaseZap RNase decontamination solution	Thermo Scientific	Cat#AM9780
Sodium deoxycholate	Sigma-Aldrich	Cat#D6750
Sucrose	Sigma-Aldrich	Cat#8510-500GM
SulfoLink coupling resin	Thermo Scientific	Cat#20402
SUPERase·In RNase inhibitor (20 U/μL)	Invitrogen	Cat#AM2696
Tris base	Fisher Scientific	Cat#BP152-500
Triton X-100	Sigma-Aldrich	Cat#T8787
Trypsin-EDTA (0.5%), no phenol red	Gibco	Cat#15400054
TURBO DNase (2 U/μL)	Invitrogen	Cat#AM2238
UltraPure DNase/RNase-free distilled water	Invitrogen	Cat#10977015
Urea	Sigma-Aldrich	Cat#U1250-1KG
Water (HPLC)	Fisher Scientific	Cat#W5-4
Experimental models: Cell lines
Mouse: E14 embryonic stem cells (E14 mESC)	17	N/A
Other
1 mL, Open-Top Thickwall polycarbonate tube	Beckman Coulter	Cat#343778
Costar Spin-X centrifuge tube filters, 0.22 μm pore cellulose acetate membrane	Corning	Cat#8160
Pierce centrifuge columns, 5 mL	Thermo Scientific	Cat#89897
ThermoMixer C	Eppendorf	Cat#5382000023
TLA120.2 rotor	Beckman Coulter	Cat#357656
TL-100 ultracentrifuge	Beckman Coulter	N/A

Materials and equipment

In general, to perform the sucrose cushion ultracentrifugation and chromatography steps, alternative reagents for buffers are acceptable as long as they are at least molecular biology-grade and RNase-free.

Cleaning the bench with RNaseZap and 70% (v/v) ethanol before beginning this protocol and using reagents and plastics set aside for RNA work is recommended to minimize RNase contamination.

Preparation of mESC media

Reagent	Final concentration	Amount
EmbryoMax ES Cell Qualified FBS	15%	75 mL
200 mM L-glutamine	2 mM	5 mL
Penicillin-Streptomycin (100×)	1×	5 mL
Non-essential amino acids (100×)	1×	5 mL
2-Mercaptoethanol	55 μM	0.5 mL
Mouse leukemia inhibitory factor (LIF)	103 U/mL	0.05 mL
KnockOut DMEM	–	409.45 mL
Total	–	500 mL

Combine the components and mix. Filter sterilize through a 0.22 μm filter. Store at 4°C in the dark for up to two weeks.

Preparation of stock solutions

1 M HEPES-KOH, pH 7.6: dissolve 119.2 g of HEPES by adding nuclease-free water to 450 mL. Adjust the pH using potassium hydroxide (KOH) pellets until it reaches pH 7.6; then add nuclease-free water to a final volume of 500 mL to make 1 M of HEPES-KOH, pH 7.6. Filter sterilize the buffer using a 0.22 μm filter. This buffer can be stored at room temperature (20°C–25°C), protected from light, for at least one year.

CRITICAL: HEPES is light sensitive, and RAPIDASH performed using HEPES with prolonged exposure to light results in lower yield. All buffers containing HEPES should be stored in the dark or wrapped in aluminum foil.

1 M Tris, pH 8.5: dissolve 60.57 g Tris in approximately 475 mL nuclease-free water. Adjust the pH using hydrochloric acid (HCl) to pH 8.5. Adjust the final volume of the buffer to 500 mL using nuclease-free water. Filter sterilize the buffer using a 0.22 μm filter. This can be stored at room temperature (20°C–25°C) for at least one year.

1 M dithiothreitol (DTT): resuspend one 7.7 mg vial of DTT in 50 μL nuclease-free water. This solution should be made fresh and stored on ice until use.

100 mg/mL cycloheximide: solubilize 50 mg cycloheximide in 100% ethanol to a final volume of 0.5 mL. This 1000× stock solution can be stored at −20°C for approximately one week. Any precipitation that is formed during storage at −20°C should be resuspended by completely warming the solution to room temperature (20°C–25°C) and vortexing rapidly.

10% (w/v) sodium deoxycholate: combine 0.05 g sodium deoxycholate with nuclease-free water up to 0.5 mL. Resuspend the solution completely either by pipetting or by repeated vortexing. This solution should be protected from light and made fresh every time.

1 M ammonium bicarbonate (NH₄HCO₃): dissolve 0.0791 g ammonium bicarbonate in a final volume of 1 mL high performance liquid chromatography (HPLC) water. Make this fresh every time.

8 M urea: dissolve 0.480 g of urea in a final volume of 1 mL HPLC water. Make this fresh every time.

Preparation of lysis buffer

Timing: 15 min

Each 15-cm plate of E14 mESCs requires at least 400 μL of lysis buffer

Reagent	Final concentration	Amount
Nuclease-free water	–	265.2 μL
1 M HEPES-KOH, pH 7.6	20 mM	8 μL
1 M magnesium acetate (Mg(OAc)2)	15 mM	6 μL
1.5 M ammonium chloride (NH4Cl)	60 mM	16 μL
1 M DTT	1 mM	0.4 μL
100 mg/mL cycloheximide	100 μg/mL	0.4 μL
10% Triton X-100	1%	40 μL
10% deoxycholate	0.5%	20 μL
100% glycerol	8%	32 μL
2 U/μL TURBO DNase	0.02 U/μL	4 μL
20 U/μL SUPERase⋅In RNase Inhibitor	0.2 U/μL	4 μL
Halt 100× protease and phosphatase inhibitor, EDTA-free	1×	4 μL
Total	–	400 μL

Combine the reagents listed in the table. DTT, cycloheximide, deoxycholate, TURBO DNase, SUPERase⋅In RNase Inhibitor, and the protease and phosphatase inhibitor must be added fresh in a 15-ml tube. Other reagents can be prepared the day before. Wrap the tube in foil and store on ice until needed.

CRITICAL: Make the buffer fresh and use it cold. Glycerol is extremely viscous, so only use a P1000 tip to pipette volumes of glycerol. For ease of preparation, make a minimum of 1.5 mL of lysis buffer.

Preparation of cushion buffer

Timing: 30 min

For each sample, 700 μL of cushion buffer is required. Cushion buffer is commonly made in a 15-mL tube by resuspending sucrose with buffer and water to a predetermined volume. For ease of preparation, make a minimum of 3 mL of cushion buffer.

Reagent	Final concentration	Amount
Sucrose (molecular weight: 342.2965 g/mol)	1 M	239.6 mg
1 M HEPES-KOH, pH 7.6	20 mM	14 μL
1 M Mg(OAc)2	15 mM	10.5 μL
1.5 M NH4Cl	60 mM	28 μL
Nuclease-free water (to:)	–	700 μL
1 M DTT	1 mM	0.7 μL
20 U/μL SUPERase⋅In RNase inhibitor	0.2 U/μL	7 μL
100 mg/mL cycloheximide	100 μg/mL	1.7 μL

Combine the HEPES-KOH, Mg(OAc)₂, NH₄Cl, and sucrose in a foil-wrapped 15-ml tube. Add water so the total volume in the tube is 700 μL and dissolve on a rocker at room temperature (20°C–25°C). Once the sucrose is dissolved, add DTT, SUPERase⋅In RNase inhibitor, and cycloheximide, mix, and store on ice until it is ready to use. Make this buffer fresh each time.

CRITICAL: Make this buffer fresh and store it on ice until use. Note that cycloheximide is toxic; use personal protective equipment when weighing the powder, such as gloves and a dust mask to minimize contact and inhalation, and dispose of the excess stock solution appropriately.

Preparation of coupling buffer

Timing: 5 min

Reagent	Final concentration	Amount
1 M Tris, pH 8.5	50 mM	25 mL
0.5 M EDTA	5 mM	5 mL
Nuclease-free water	–	470 mL
Total	–	500 mL

Combine the reagents. Filter sterilize with a 0.22 μm filter. Store at 4°C, where it is stable for at least 6 months.

Preparation of L-cysteine-containing coupling buffer

Timing: 5 min

Reagent	Final concentration	Amount
L-cysteine (molecular weight: 121.16 g/mol)	50 mM	12.116 mg
Coupling buffer	–	2 mL

Dissolve L-cysteine in coupling buffer in a foil-wrapped tube. Make this buffer fresh every time.

CRITICAL: Make this buffer fresh and protect it from light. L-cysteine in solution can oxidize to form cystine, and this is accelerated when exposed to light and high pH.

Preparation of priming buffer and binding buffer

Timing: 10 min

Priming buffer

Reagent	Final concentration	Amount
1 M HEPES-KOH, pH 7.6	20 mM	420 μL
1 M Mg(OAc)2	15 mM	315 μL
1.5 M NH4Cl	60 mM	840 μL
1 M DTT	1 mM	21 μL
100 mg/mL cycloheximide	100 μg/mL	21 μL
Remove for binding buffer	–	77 μL
Nuclease-free water	–	18,460 μL
Total	–	20,000 μL

Binding buffer

Reagent	Final concentration	Amount
1 M HEPES-KOH, pH 7.6	20 mM	20 μL
1 M Mg(OAc)2	15 mM	15 μL
1.5 M NH4Cl	60 mM	40 μL
1 M DTT	1 mM	1 μL
100 mg/mL cycloheximide	100 μg/mL	1 μL
Add from priming buffer:	–	77 μL
Nuclease-free water	–	903 μL
2 U/μL TURBO DNase	20 U/mL	10 μL
20 U/μL SUPERase⋅In RNase Inhibitor	200 U/mL	10 μL
Total	–	1000 μL

Combine the HEPES-KOH, Mg(OAc)₂, NH₄Cl, DTT, and cycloheximide to make the priming buffer master mix in a foil-wrapped 15-ml tube. Remove 77 μL of this master mix into a separate foil-wrapped 15-ml tube for the binding buffer.

• •To complete the priming buffer, add nuclease-free water. Leave the foil-wrapped tube on ice.
• •To complete the binding buffer, add TURBO DNase, SUPERase⋅In RNase Inhibitor, and nuclease-free water to the 77 μL priming buffer master mix that had been set aside. Leave the foil-wrapped tube on ice.

CRITICAL: Prepare the priming and binding buffers fresh and protect them from light.

Note that any alterations to the lysis, binding, and priming buffers can affect which RAPs are identified, as RAP interaction with the ribosome can be salt-sensitive.

Preparation of elution buffer

Timing: 5 min

Reagent	Final concentration	Amount
Nuclease-free water	–	713 μL
1 M HEPES-KOH, pH 7.6	20 mM	20 μL
1 M Mg(OAc)2	15 mM	15 μL
2 M KCl	500 mM	250 μL
1 M DTT	1 mM	1 μL
100 mg/mL cycloheximide	100 μg/mL	1 μL
Total	–	1000 μL

Combine the reagents above in a foil-wrapped 15-ml tube. Store on ice.

CRITICAL: Elution buffer must be prepared fresh and protected from light.

Preparation of the urea buffer for mass spectrometry processing

Reagent	Final concentration	Amount
HPLC water	–	120 μL
1 M NH4HCO3	50 mM	30 μL
8 M urea	6 M	450 μL
Total	–	600 μL

Combine the reagents above. Make this buffer fresh every time.

Preparation of the ultracentrifuge and other centrifuges

Timing: approximately 1 h

• •Chill the TLA120.2 rotor in the cold room (at 4°C) or inside the TL-100 centrifuge.
• •Set the TL-100 ultracentrifuge to 100,000 rpm, 1 h, 4°C to begin chilling.
• •Chill all benchtop centrifuges and microcentrifuges to 4°C except for the benchtop centrifuge that will be used at room temperature during the preparation of the sulfhydryl-charged resin.

Step-by-step method details

Cell harvest and lysis

Timing: 1.5 h

In this step, cycloheximide is used to stabilize the polysomes prior and throughout the harvest and lysis. The cells are pelleted, and cytoplasmic lysate is generated.

• 1.
  Prepare solutions to harvest the cells. Warm mESC media to replace the spent media in the culture dishes using a 37°C water bath. Prepare at least 18 mL for one 15-cm plate.

  • a.
    Aliquot enough media to make 10× cycloheximide (1 mg/mL cycloheximide in mESC media) into a 50 mL tube.

    • i.
      Prepare at least 2 mL for one 15-cm plate.
    • ii.
      Warm this media in a 37°C water bath.
  • b.
    Aliquot enough media to make 1× cycloheximide (100 μg/mL cycloheximide in mESC media) to neutralize trypsin into 50 mL tubes.

    • i.
      Prepare at least 8 mL for one 15-cm plate.
    • ii.
      Leave this in a 4°C fridge.
  • c.
    Aliquot at least 20 mL for one 15-cm plate of DPBS into tubes, and warm the DPBS in a 37°C water bath.
  • d.
    Aliquot at least 4 mL for one 15-cm plate of 0.05% trypsin-EDTA, and warm this in a 37°C water bath.
  • e.
    Aliquot at least 3 mL DPBS per sample for cell pellet resuspension. Keep it in a 4°C fridge.
• 2.Approximately 45 min before harvesting the cells, replace the spent media in the 15-cm plate with 18 mL warm mESC media.

Note: This step helps to stimulate translation and generate more polysomes. This works well for mESCs but may not be applicable to other sample types.

• 3.
  Remove the warmed media aliquot from step 1a from 37°C water bath and add cycloheximide to make a 10× (1 mg/mL cycloheximide) solution.

  • a.
    Add enough 10× cycloheximide in media to the cells such that the final concentration is 100 μg/mL of cycloheximide, e.g., add 2 mL 10× cycloheximide to one 15-cm plate.
  • b.
    To do this, add the 10× cycloheximide dropwise over the plate and then shake the plate to disperse the cycloheximide.
• 4.Place the cells back into the tissue culture incubator for 3 min.
• 5.Remove the DPBS aliquot from step 1c and the 0.05% trypsin-EDTA aliquot from step 1d from the water bath. Add cycloheximide to each solution to a final concentration of 100 μg/mL cycloheximide.
• 6.Add cycloheximide to a final concentration of 100 μg/mL cycloheximide in the cold media aliquot for neutralizing trypsin (from step 1b).
• 7.After the 3-min incubation, remove the cells from the incubator and wash them with 10 mL warm 100 μg/mL cycloheximide in DPBS (from step 5) to the 15-cm plate twice.
• 8.Add 4 mL 100 μg/mL cycloheximide in 0.05% trypsin-EDTA (from step 5) to the 15-cm plate and return the cells to the incubator for around 5 min, or until the cells detach.
• 9.Neutralize the trypsin with at least 4 mL cold mESC media with 100 μg/mL cycloheximide (prepared in step 6).
• 10.Collect the cells into a 15-mL conical (or any suitable tube that can be centrifuged) and centrifuge the sample at 4°C, 200 × g for 3 min using a benchtop centrifuge.
• 11.Add cycloheximide to the aliquoted DPBS for resuspension (from step 1e) to a final concentration of 100 μg/mL.
• 12.Aspirate the supernatant and wash the cells by resuspending the cell pellet in 2 mL cold 100 μg/mL cycloheximide in DPBS (from step 11).
• 13.Centrifuge the sample at 4°C, 200 × g for 3 min using a benchtop centrifuge.
• 14.Aspirate the supernatant and resuspend the cell pellet in 1 mL cold 100 μg/mL cycloheximide in DPBS from step 11. Transfer the sample to pre-chilled Eppendorf tubes.
• 15.Centrifuge the sample at 4°C, 200 × g for 3 min using a benchtop microcentrifuge.
• 16.Remove the supernatant with a micropipette. Briefly spin the sample and remove any remaining supernatant with a P10 micropipette.

Note: The cell pellet can be flash frozen and stored at −80°C.

• 17.Add 400 μL of chilled lysis buffer to the mESC pellet derived from one 15-cm plate (approximately 40–50 × 10⁶ cells).
• 18.Vortex the sample for 30 s and then place the sample back on ice for 30 s. Repeat this process two more times.
• 19.Incubate the sample on ice for 30 min. During this incubation, vortex the sample for 10 s every 10 min.
• 20.
  Clarify the sample by performing a series of centrifugations at 4°C and moving the supernatant to a fresh, chilled tube each time.

  • a.
    First, remove nuclei and cell debris with two consecutive centrifugations at 800 × g for 5 min.
  • b.
    Then, do one centrifugation at 8,000 × g for 5 min.
  • c.
    Finally, do one centrifugation at 20,817 × g (max speed) for 5 min.

Sucrose cushion ultracentrifugation

Timing: 1.5 h

This step pellets large, dense complexes present in the cytoplasmic lysate.

• 21.
  Add 700 μL cushion buffer into an ultracentrifugation tube.

  • a.
    Use P200 tips to layer 300 μL clarified cytoplasmic lysate on top of the cushion buffer.
  • b.
    To do this, gently dispense the lysate against the wall of the ultracentrifugation tube to minimally disturb the sucrose cushion buffer.
  • c.
    Leftover cytoplasmic lysate can be flash frozen in liquid nitrogen and stored at −80°C to analyze the input sample by western blotting or other readouts.
• 22.Centrifuge the sample at 100,000 rpm for 1 h at 4°C.
• 23.Remove the supernatant by pipetting.
• 24.Resuspend the cushion pellet with the 1 mL of binding buffer. If desired, some cushion pellet sample can be retained for analysis by western blotting.

Preparation of sulfhydryl-charged SulfoLink resin

Timing: 1.5 h

This step, in which cysteine is coupled to the SulfoLink resin, is performed while the samples are undergoing sucrose cushion ultracentrifugation.

• 25.
  Pipette 50% SulfoLink resin slurry into a 15-mL tube that can be centrifuged, such as a 15-mL conical. The resin capacity for RNA binding is ˜20 A260 units per mL.

  • a.
    For each cushion pellet from a 15-cm plate of mESCs, use 2 mL 50% SulfoLink resin slurry, which is approximately equal to 1 mL SulfoLink resin bed volume (BV).
  • b.
    If scaling up for multiple samples, a maximum of 5 mL of 50% SulfoLink resin slurry should be added to a single 15-mL tube.
  • c.
    Centrifuge at 850 × g for 1 min at room temperature (20°C–25°C) and remove the supernatant.
• 26.
  Wash the resin three times in coupling buffer by resuspending the resin in 2 volumes of coupling buffer : 1 volume SulfoLink resin BV i.e., 2 mL coupling buffer for 1 mL SulfoLink resin BV, and inverting the tube several times.

  • a.
    Centrifuge the tube at 850 × g for 1 min at room temperature (20°C–25°C).
  • b.
    Pour out the supernatant.
• 27.Resuspend the 1 volume of SulfoLink resin BV with 2 volumes of 50 mM solution of L-cysteine in coupling buffer. Wrap the tube in foil.
• 28.Rock or rotate the tube of slurry for at least 1 h at room temperature (20°C–25°C).

Note: This step can go up to 2 h.

• 29.
  Centrifuge the tube at 850 × g for 1 min at room temperature (20°C–25°C).

  • a.
    Pour out the supernatant and resuspend the resin in coupling buffer as described in step 26.
  • b.
    Repeat this step two more times for a total of three washes.
• 30.Equilibrate the resin by washing it three times with 4 volumes of priming buffer for 1 volume of SulfoLink resin BV (e.g., 4 mL priming buffer for 2 mL 50% SulfoLink slurry, which is approximately equal to 1 mL of SulfoLink resin BV) as described in step 26.
• 31.Resuspend the resin in 2 volumes of priming buffer : 1 volume of SulfoLink resin BV.
• 32.
  Remove the twist-off bottom of a 5 mL Pierce centrifuge column and reseal the column with a press-on cap. Use a serological pipette to transfer the resin suspension into the column.

  • a.
    If preparing more than one column, do one column at a time and resuspend the resin by pipetting prior to each transfer to prevent the resin from settling.
  • b.
    Cap the top of the column, place it into a conical tube, cover the tube with foil, and keep it on ice or in a 4°C fridge.

Chromatographic enrichment of ribosomes and RAPs

Timing: 1.5 h

During this step, RNA-containing protein complexes, which are mostly ribosomes and RAPs, are enriched from the sucrose cushion pellet sample.

• 33.Immediately before use, remove the bottom cap, place the column into a conical tube, and pellet the resin by centrifuging for 1 min at 1,000 × g at 4°C to remove the storage solution. Then, cap the bottom of the column.
• 34.
  Add 1 mL of resuspended cushion pellet per 1 mL of SulfoLink resin BV. Typically, this corresponds to 50 – 100 μg RNA for mESCs, although we have used as little as 20 μg RNA in the case of mouse embryonic tissues.

  • a.
    If desired, retain 10 - 20 μg protein from the cushion pellet sample for analysis by western blotting.
  • b.
    Cap the top of the column and mix by tapping the column.
  • c.
    Place the column into the conical tube and incubate on ice without tumbling for 15 min.
• 35.
  Remove the bottom cap of the column and place the column into a new conical tube.

  • a.
    Centrifuge at 1,000 × g at 4°C for 1 min.
  • b.
    Cap the bottom of the column, place the flowthrough fraction back into the column, and repeat steps 34b and 34c.
• 36.
  Remove the bottom cap and place the column back into the conical tube.

  • a.
    Centrifuge the sample at 1,000 × g at 4°C for 1 min.
  • b.
    If desired, flash freeze the flowthrough sample in liquid nitrogen and store it at −80°C for analysis later.
• 37.
  Cap the bottom of the column and add 2 volumes of priming buffer : 1 volume SulfoLink resin BV.

  • a.
    Cap the top of the column and mix the column by inverting by hand until the resin is resuspended.
  • b.
    Remove the bottom cap and place the column into a new conical tube.
  • c.
    Centrifuge the column at 1,000 × g at 4°C for 1 min.
  • d.
    Repeat this washing protocol three more times for a total of 4 washes.
  • e.
    Flash freeze the washing solution in liquid nitrogen and store it at −80°C for troubleshooting purposes.
• 38.
  To elute the ribosomes and RAPs, cap the bottom of the column, add 500 μL elution buffer per 1 ml of SulfoLink resin BV, and cap the top of the column.

  • a.
    Mix by inverting the column and place the column into the conical tube.
  • b.
    Incubate the tube on ice for 2 min.
• 39.
  Remove the bottom cap and place the column into a new conical tube.

  • a.
    Centrifuge at 1,000 × g at 4°C for 1 min. Collect the eluate.
  • b.
    Repeat step 38 and combine the eluates.

Note: The eluate can be flash frozen in liquid nitrogen and stored at −80°C.

• 40.
  Precipitate the protein using a kit such as the ProteoExtract Protein Precipitation kit following the manufacturer’s instructions.

  • a.
    If the downstream readout is western blotting, resuspend the protein pellet using 50 μL 1× Laemmli buffer.

    • i.
      Boil at 95°C for 10 min while shaking at 1,000 rpm on the ThermoMixer to denature the proteins.
    • ii.
      Spin down the tubes briefly.
    • iii.
      This can then be stored at −20°C.
  • b.
    If the downstream readout is mass spectrometry, then the protein pellet can be resuspended in 50 μL 6 M urea in 50 mM NH₄HCO₃ to denature the proteins for subsequent processing for mass spectrometry. While this sample should be compatible with many workflows that process proteins for mass spectrometry, this method of resuspending the sample in 6 M urea allows for the complete dissolution of the protein pellet.

Expected outcomes

One 15-cm plate of mESCs should yield approximately 250 μg of protein, which includes the core ribosomes and RAPs. When the RNA of the RAPIDASH eluate is analyzed, there should be clear bands indicating that the 28S and 18S rRNA species were isolated as intact rRNAs (Figure 2A). The cytoplasmic lysate, sucrose cushion pellet, and RAPIDASH eluate samples can be analyzed by western blotting for presence of contaminants such as nuclear pore, cytoskeletal, and mitochondrial proteins that are present in the sucrose cushion pellet sample but should be depleted in the RAPIDASH (Figure 2B). Similarly, these samples should show that known RAPs remain in the RAPIDASH eluate sample (Figure 2C). Note that the RAPs will not necessarily appear enriched in the RAPIDASH eluate compared to the cytoplasmic lysate or sucrose cushion samples because RAPs can have extra-ribosomal populations, such that only a small pool of a given RAP is actually bound to ribosomes.^1,6 The RAPIDASH eluate can then be processed further for mass spectrometry-based proteomics to identify candidate RAPs in the sample.

Figure 2.

Expected outcomes for a successful RAPIDASH experiment

(A) RNA electropherogram output from an Agilent 2100 bioanalyzer of E14 mESC sucrose cushion pellet (top) and RAPIDASH eluate (bottom) samples, which is used to assess ribosomal RNA (rRNA) integrity. Mouse 18S rRNA is approximately 1,900 nucleotides (nt), and mouse 28S rRNA is approximately 4,700 nt. RIN stands for RNA integrity number, which assigns a value between 1 to 10 to an RNA electropherogram trace, with 10 indicating minimal degradation.

(B) Western blotting of E14 mESC cytoplasmic lysate, sucrose cushion pellet, and RAPIDASH eluate samples to evaluate the specificity of RAPIDASH for ribosomes over non-ribosomal complexes. Approximately equal amounts of ribosomes for the sucrose cushion pellet and RAPIDASH eluate samples were analyzed by western blotting for Nup62, Atp5a1, and Tom20, components of the nuclear pore complex, ATP synthase, and the translocase of the outer membrane (TOM) complex, respectively. Rpl4, Rpl29, Rps5, Rps26, and Rps27 are ribosomal proteins. Cytoplasmic lysate was included as an input control. Molecular weight markers are in kilodaltons (kDa). Figure reprinted with permission from Susanto et al., 2024.¹

(C) Western blotting of RAPIDASH eluate for the presence of known ribosome-associated proteins (RAPs). To assess the ability of RAPIDASH to retain known RAPs in the RAPIDASH eluate, 1% of the E14 mESC cytoplasmic lysate volume and 35% of the RAPIDASH eluate volume were probed by western blotting for the presence of the known RAPs Metap1, Ufl1, Upf1, Ddx1, and Nsun2. Molecular weight markers are in kDa. Figure reprinted with permission from Susanto et al., 2024.¹

(D and E) Sucrose gradient fractionation of E14 mESC cytoplasmic lysate and western blotting for the RAP Dhx30. E14 mESC cytoplasmic lysate was fractionated using sucrose gradients without (D) or with (E) EDTA. The proteins in each fraction were precipitated, and equal volumes of protein sample were loaded and analyzed by western blotting for the presence of Dhx30 and for the ribosomal protein markers Rps26 and Rpl29. Molecular weight markers are in kDa. Figure reprinted with permission from Susanto et al., 2024.¹

Limitations

While RAPIDASH is a facile technique to generate a list of candidate RAPs from any biological sample, there are some limitations. The precise mechanism that governs the affinity of RNA to the sulfhydryl-charged resin is unclear; therefore, we have empirically optimized and shown that this chromatography step can enrich RAP-containing ribosomes while depleting other dense protein complexes present in sucrose cushion pellet samples that do not contain RNA. While the sulfhydryl-charged resin has an affinity for RNA, in our conditions, poly(A)-enriched RNA does not bind the sulfhydryl-charged resin, which suggests the mechanism of enrichment relies on an RNA feature (e.g., structure or modification) that is not present in most poly(A)-enriched RNA. As the ribosome is an extremely abundant ribonucleoprotein (RNP) complex, it is highly enriched during the chromatography step and is the main, but not exclusive, RNP complex in the final sample. The buffer and centrifugation conditions described here were optimized to maintain the interactions between ribosomes and RAPs in mammalian samples. It is possible that other samples may require slightly different conditions, particularly during the lysis step. When applying RAPIDASH to other sample types, blotting for markers of dense, non-ribosomal complexes such as the nuclear pore complex to ensure they are depleted in the RAPIDASH eluate compared to the sucrose cushion pellet sample, and blotting the RAPIDASH eluate for bona fide RAPs to ensure they are still found in the final sample, can reveal whether steps need to be optimized. If buffer compositions need to be adjusted, it is important to remember that RNA binds to the sulfhydryl-charged resin in buffers with low salt concentrations and elutes in buffers with high salt concentrations.

Once candidate RAPs have been identified, they must be tested by orthogonal techniques to verify their association to the ribosome, such as sucrose gradient fractionation with and without EDTA, which causes ribosomes to dissociate into the respective small and large subunits and thus shift into lighter fractions of the sucrose gradient. RAPs should co-migrate with the ribosomal subunits (Figures 2D and 2E). Additional limitations include the fact that the lower limit of input needed to generate enough RAPIDASH eluate for mass spectrometry-based proteomic analysis has not been fully tested, which may prohibit the analysis of samples with extremely limited material. Finally, RAPs often have extra-ribosomal pools in cells; thus, RAPs must be carefully studied to distinguish their ribosomal and extra-ribosomal functions.

Troubleshooting

Problem 1

The ribosomes are degrading during the procedure as visualized by rRNA degradation according to the Bioanalyzer.

Potential solution

Remake reagents and ensure they are RNase-free.

Problem 2

Ribosomes are not binding to the sulfhydryl-charged resin.

Potential solution

This chromatography works by binding in low salt conditions and eluting in high salt conditions. Any adjustments to the binding and elution buffers must ensure the binding buffer has a low salt concentration. In addition, heparin interferes with the binding of ribosomes onto the resin.

Problem 3

The yield is low.

Potential solution

Low yield is observed if HEPES-containing buffers were exposed to light or if the cysteine-charged resin dried out during the experiment. Remake HEPES-containing reagents and wrap the containers in foil to protect them from light. Work quickly and stagger samples to avoid having the resin dry out (steps 25–39).

Problem 4

The sucrose cushion pellet does not resuspend well (step 24).

Potential solution

To help resuspend the sucrose cushion pellet, add the priming buffer to the centrifuge tube containing the sucrose cushion pellet, parafilm the top of the tube, and shake the tube in the cold room (at 4°C) on a thermal mixer for 30 min.

Resource availability

Lead contact

Requests for further information should be directed to the lead contact, Maria Barna (mbarna@stanford.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Victoria Hung (vhung3@stanford.edu) and Teodorus Theo Susanto (susantott@a-star.edu.sg).

Materials availability

This manuscript did not generate new unique reagents.

Data and code availability

Original data used for this manuscript are available from the lead contact upon request.

Acknowledgments

This work was funded by National Institutes of Health (NIH) grants 5R21MH130323 and 5R01HD086634 (to M.B.). V.H. was the Fraternal Order of Eagles Fellow of the Damon Runyon Cancer Research Foundation (DRG 2314-17) and was also supported by the Katharine McCormick Advanced Postdoctoral Fellowship from Stanford University, School of Medicine. T.T.S. was supported by a National Science Scholarship (PhD) from the Agency for Science, Technology, and Research.

Author contributions

V.H., T.T.S., and M.B. conceptualized and developed the technique. T.T.S. acquired the data. V.H. and T.T.S. wrote the manuscript. V.H., T.T.S., and M.B. edited the manuscript.

Declaration of interests

The authors declare no competing interests.