Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners

Materials and Reagents

1. 150 mm tissue culture plate (TPP, catalogue number: 93150 )
2. Cell scrapers (TPP, catalog number: 99003 )
3. Falcon tubes (TPP catalog number: 91015 )
   
4. Surgical Blade (Swann-Morton, catalog number: 0 103 )
5. 96 Deepwell Plate (Thermo, catalog number: 95040450 )
6. 2 ml Protein LoBind Tubes (Eppendorf, catalog number: 00 30108132 )
7. 1.5 ml Protein LoBind Tubes (Eppendorf, catalog number: Z666505 )
8. 0.5 ml Protein LoBind Tubes (Eppendorf, catalog number: 00 30108094 )
9. 12 Channel Pipette, 30-300 μl (Eppendorf, catalog number: 3122000060 )
10. 10% NP-40 (Thermo Fischer Scientific, catalog number: 85124 )
11. PBS (Gibco, catalog number: 10010-015 )
12. Glutaraldehyde (Merk, catalog number: 111-30-8 )
13. Glycine (Fluka, catalog number: 15527H )
14. Protease inhibitors cOmplete, Mini, EDTA-free (Roche, catalog number: 0 4693159001 )
15. DTT (Merck, catalog number: 3483-12-3)
16. Superase-In (Ambion, catalog number: AM2694 )
17. Stellaris biotinylated probes (custom made by Biosearch as per gene of interest)
18. MagReSyn^® Streptavidin Affinity binding/capture of biotinylated biomolecules (AEC-AMERSHAM SOC Ltd, catalog number: MR-STV002 )
19. Precision Plus Protein Dual Color Standards (Bio-Rad, catalog number: 161-0374 )
20. 4x Laemmli sample buffer (Bio-Rad, catalog number: 1610747 )
    
21. Coomassie Brilliant Blue R-250 (Bio-Rad, catalog number: 1610436 )
22. Bovine Serum Albumin Fraction V (Roche, catalog number: 10735078001 )
23. LC-MS Water (Honeywell, catalog number: LC365 )
24. Ammonium Bicarbonate (Fluka, catalog number: 0 9830 )
25. Methanol (Honeywell, catalog number: LC230 )
26. Acetonitrile (Honeywell, catalog number: LC015 )
27. Iodoacetamide (Sigma-Aldrich, catalog number: I1149 )
28. Trypsin (Promega, catalog number: V511A )
29. Formic Acid (Thermo, catalog number: 28905 )
30. Tris-HCl (Sigma-Aldrich, catalog number: T1503-250G )
31. KCl (Sigma-Aldrich, catalog number: P9333-1KG )
32. Triton X100 (Sigma-Aldrich, catalog number: X-100-100ML )
33. NaCl (Sigma-Aldrich, catalog number: s7653-1KG )
34. SDS (Sigma-Aldrich, catalog number: L3771-100G )
35. Formamide (Thermo Fischer Scientific, catalog number: 17899 )
36. 20x SSC (Sigma-Aldrich, catalog number: S6639-1L )
37. RNase A (Sigma-Aldrich, catalog number: 10109142001 )
38. RNase H (Sigma-Aldrich, catalog number: 10786357001 )
39. DNase 1 (Invitrogen, catalog number: EN0521 )
    
40. Lysis Buffer (see Recipes)
41. Hybridization Buffer (see Recipes)
42. Washing Buffer (see Recipes)
43. DNase Buffer (see Recipes)
44. Elution Buffer (see Recipes)
45. Destaining Buffer (see Recipes)
46. Dehydration Buffer 1 (see Recipes)
47. Reduction Buffer (see Recipes)
48. Alkylation Buffer (see Recipes)
49. Dehydration Buffer 2 (see Recipes)
50. Digestion Buffer (see Recipes)
51. Trypsin Solution (see Recipes)
52. Extraction Buffer (see Recipes)
53. Loading Solvent (see Recipes)
54. Gradient solvent A (see Recipes)
55. Gradient solvent B (see Recipes)
    

Equipment

1. Refrigerated microcentrifuge (Beckman Coulter, model: 22R )
2. Shaker (TCS Scientific Corp Nutator)
3. Rotator (Intelli-mixer RM-2L)
4. Magnetic separator (Resyn Biosciences)
5. Deepwell Plate Thermal Mixer (Eppendorf, catalog number: 5355 )
6. Deepwell Plate Vacuum Drying System (Gel plugs dried in vacuum desiccator jar trough Agilent’s DS 102 dual stage rotary vane pump, catalog number: 9499315 )
7. Vacuum Concentrator (Labconco, catalog number: 7310035 )
8. Water Bath Sonicator (Bandelin, catalog number: 301 )
9. Dionex Ultimate 3000 RSLC System (Thermo, catalog number: IQLAAAGABHFAPBMBEZ )
10. Desalting Column (Thermo, catalog number: 164567 )
11. Separation Column (Thermo, catalog number: 164534 )
    

Software

1. Protein Pilot (Sciex, https://sciex.com/products/software/proteinpilot-software)

Procedure

1. Crosslinking of cells
   1. Grow ~1 x 10⁷ cells in desired conditions in 150 mm plates (in a volume of 30 ml medium).
      Note: Higher number of cells can be used, we found 1 x 10⁷ cells to be the minimal amount required to obtain data at high resolution.
   2. Wash in 20 ml 1x PBS.
   3. Crosslink cells in the plate with 20 ml 1% Glutaraldehyde in 1x PBS for 10 min at RT on a shaker.
      Note: Alternative cross linkers can be used instead of Glutaraldehyde, including Ultraviolet light and Formaldehyde. The protocol has to be adjusted accordingly.
   4. Quench with 20 ml 0.125 M Glycine for 5 min at RT, on a shaker.
   5. Wash with 20 ml ice-cold 1x PBS.
   6. Scrape cells and transfer to 15 ml falcon tubes.
   7. Centrifuge cells at 2,500 x g, 5 min, at 4 °C and discard supernatant.
   8. Snap freeze pellet and store at -80 °C or use immediately.
      
      
2. Lysis of cells
   1. Resuspend pellet in 250-300 μl Lysis buffer + 1x protease Inhibitors.
   2. Transfer the re-suspended cells to an Eppendorf, mix well and incubate at RT for 25 min on a shaker.
   3. Centrifuge cells at 4 °C for 10 min at 10,000 x g.
   4. Transfer the supernatant to a new Eppendorf and discard pellet.
   5. Centrifuge supernatant to pre-clear, at 4 °C for 10 min at 12,000-14,000 x g.
   6. Collect the supernatant (the pellet is not always visible at this stage).
      
      
3. Probe hybridization
   1. Prepare the hybridization reaction:
      200 μl lysate
      100 μl Hybridization buffer
      0.3 μl 1 M DTT (to a final of 0.1 mM)
      1x Protease inhibitors
      1.5 μl 20 U/μl Superase (to a final of 0.1 U/μl)
      Add biotinylated probes to a final concentration of 100 pmol per reaction.
      Note: We recommend the use of a set of scrambled biotinylated single molecule probes as a negative control. Alternatively, a set of probes that target the antisense strand can be used. Additionally, we recommend a parallel reaction, were the set of probes are omitted. Data sets of the experiment should be compared with these control datasets to eliminate proteins that tend to aggregate or bind in a non-specific manner.
   2. Incubate hybridization reaction at 37 °C for 4 h, rotating.
      
      
4. Streptavidin beads preparation (Video 1)
   Note: In the video the tube in which the reaction will occur is labeled “Reaction”. Subsequently, there are three tubes, labeled “1x PBS”, “1 mg/ml BSA” and “Hyb” (hybridization buffer).
   1. Wash 40 μl slurry Streptavidin beads (equals to ~10-15 μl solid Streptavidin beads) in ~250 μl 1x PBS x 3.
   2. Block beads with 250-500 μl 1 mg/ml BSA in 1x PBS for 1 h at RT, shaking.
   3. Discard the blocking solution and wash beads with Hybridization buffer. Repeat wash three times.
      
      
      
      Video 1. Streptavidin beads preparation
      
      
5. Biotin-Streptavidin reaction
   1. Add the hybridization reaction to beads and incubate at 37 °C for 30-60 min, rotating.
   2. Discard the unbound reaction.
   3. Wash beads with 40x solid bead volume (~400-600 μl) of washing buffer.
   4. Repeat wash three times.
      
      
   Elution Option I:
   1. Resuspend beads in 3x the original volume of solid beads (~30-45 μl) of DNase buffer.
   2. Add 100 μl Elution buffer and incubate at 37 °C for 30 min, rotating.
   3. Add Sample Loading Buffer and boil at 95 °C for 5 min.
      
      
      Figure 1. Schematic of biochemical pulldown of RNAs and associated proteins and characterization by LS-MSMS
      
   
   Elution Option II:
   1. Add ~50 μl 2x Sample Loading Buffer and boil at 95 °C for 5 min.
   2. Run samples on a SDS-PAGE page, stain with Coomassie and cut bands for mass-spectrometry. A representative SDS-PAGE gel is shown in Figure 2.
      
      
      Figure 2. A representative image of an SDS-PAGE stained with Coomassie representing a pulldown experiment in 3T3 mouse fibroblasts: a set of probes against an mRNA of interest were used. The negative control consisted of a set of scrambled, non-specific probes
      
      
6. In-gel digestion (based on Reference 19)
   1. Cut relevant gel lane with surgical blade into multiple slices (about 8 horizontal slices along length of lane in this case, large high intensity protein bands were cut out separately to minimize potential masking of low abundance proteins).
   2. Dice each gel slice into small pieces (1 x 1 mm) and place into deepwell plate.
   3. Add ~200 μl (or enough to cover) of Destaining buffer and vortex (800 rpm max.) in a thermomixer for 20 min and discard the supernatant.
   4. Repeat Step F3.
   5. Add 100 μl (or enough to cover) of Dehydration buffer 1, vortex in a thermomixer for 20 min (800 rpm max).
   6. Remove and discard the supernatant.
   7. Vacuum the gel pieces to complete dryness (10-20 min).
   8. Add 25 μl (or enough to cover gel pieces) Reduction buffer to dried gels.
   9. Vortex and spin briefly (800 rpm max).
   10. Allow reaction to proceed at 60 °C for 1 h.
   11. Chill tubes to room temp (22-23 °C).
   12. Add 500 μl Dehydration buffer 1.
   13. Incubate for 10 min (22-23 °C).
   14. Remove supernatant and discard.
   15. Add 25 μl (or enough to cover gel pieces) Alkylation buffer to the gel pieces.
   16. Vortex and spin briefly (800 rpm max).
   17. Allow reaction to proceed in the dark for 20 min at room temperature (22-23 °C).
   18. Remove supernatant and discard.
   19. Wash gels with 100 μl Digestion buffer, vortex 10 min, spin.
   20. Remove supernatant and discard.
   21. Dehydrate gels with 100 μl (or enough to cover gel pieces) of Dehydration buffer 2, vortex 5 min, spin, remove supernatant and discard (800 rpm max).
   22. Repeat Step F21.
   23. Vacuum the gel pieces to complete dryness (~20 min, keep time to minimum).
   24. Place the deepwell plate on ice.
   25. Add freshly prepared trypsin solution to just barely cover the gel pieces (~5-50 μl).
   26. After 30 min check if all trypsin solution has absorbed and add more if necessary. (Gel pieces should be covered completely with trypsin).
   27. Rehydrate the gel pieces on ice or at 4 °C for further 1 h.
   28. Add Digestion buffer as needed to cover the gel pieces.
   29. Incubate at 37 °C for 18 h.
   30. Transfer the digest solution (aqueous extraction) into 2 ml Protein Lo-Bind tubes.
   31. To the gel pieces, add 30 μl (enough to cover) of Extraction buffer.
   32. Vortex 20-30 min.
   33. Sonicate in ultrasonic bath for 5 min.
   34. Add aqueous extraction to solution obtained at Step F30.
   35. Repeat Steps F31-F33.
   36. Speed Vac the digest solution to dryness.
       
       
7. LC-MSMS
   1. Add 20 μl loading solvent to dried peptide digests and vortex for 1 min.
   2. Transfer 18 μl to glass HPLC vial or 0.5 ml Eppendorf Protein LoBind tube and place in autosampler.
   3. Inject 2-5 μl on the trap column (PepMap 75 μm x 2 cm) at 5 μl/min using Loading solvent and after 5 min switch inline with analytical column (PepMap 75 μm x 15 cm).
   4. Elute peptides using a 15 min linear gradient 6-35% Gradient solvent B at 300 nl/min.
   5. Apply an electrospray voltage of 2.0-2.5 kV to nanospary emitter.
   6. Acquire data in Data Dependant Acquisition (DDA) mode with precursor ions acquired using a single 250 ms scan in the range 400-1,500 m/z and 50 product ion scans at 50 ms scan time in the range 100-1,800 m/z that are automatically trigger.

Data analysis

Data was processed using Protein pilot v5.0.1 using Paragon search engine (Sciex) using a Uniprot Swissprot Mouse database supplemented with sequences of common contaminating proteins. A 1% FDR cut-off was applied on PSM, peptide and proteins levels. Only proteins that were not present in the control sample lists were selected for further analysis.

Recipes

1. Lysis Buffer
   50 mM Tris-HCl pH 7.5
   150 mM KCl
   1 mM EDTA
   0.5 % Triton X100
2. Hybridization Buffer
   500 mM NaCl
   1% SDS
   100 mM Tris-HCl, pH 7.0
   10 mM EDTA
   15 % Formamide
   Add fresh before use: 1 mM DTT, Protease Inhibitors, U/μl Superase
3. Washing Buffer
   2x SSC
   0.5% SDS
   Add fresh 1 mM DTT
4. DNase Buffer
   10 mM NaCl
   0.1% NP-40
5. Elution Buffer
   100 μg/ml RNase A
   0.1 U/μl RNase H
   100 U/ml DNase 1
   All In-Gel Digestion Buffers below are prepared with LC-MS Water
6. Destaining Buffer
   50% Methanol
   50 mM Ammonium Bicarbonate
7. Dehydration Buffer 1
   100% Acetonitrile
8. Reduction Buffer
   10 mM Dithiothreitol
   25 mM Ammonium Bicarbonate
9. Alkylation Buffer
   55 mM Iodoacetamide
   25 mM Ammonium Bicarbonate
10. Dehydration Buffer 2
    50% Acetonitrile
    25 mM Ammonium Bicarbonate
11. Digestion Buffer
    25 mM Ammonium Bicarbonate
12. Trypsin Solution
    10 ng/μl Trypsin
    25 mM Ammonium Bicarbonate
13. Extraction Buffer
    50% Acetonitrile
    5% Formic Acid
14. Loading Solvent
    2% Acetonitrile
    0.2% Formic Acid
15. Gradient solvent A
    0.1% Formic acid
16. Gradient solvent B
    80% Acetonitrile
    0.1% Formic acid
    

Acknowledgments

We thank Dr. Sam Barichievy and Dr. Stephanie Fanucchi for stimulating and fruitful discussions. We also thank Dr. Nicolas Beaume for his assistance with the making of the video. This research has been supported by the following grants: V2YGE81 to A.F.S., PG-V2KYPO7, TA 2011 011 from the Council for Industrial and Scientific Research (CSIR, South Africa) to M.M.M and by a grant from the Emerging Research Area Program of The Department of Science and Technology (DST, South Africa) Department of Science & Technology Centre of Competence Grant, SA Medical Research Council SHIP grant, CSIR Parliamentary Grant.

Competing interests

The authors declare no competing interests.

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