CXMS analysis of model proteins

Enrichment of Cross-linked Peptides Using Leiker

Dan Tan and Meng-Qiu Dong

Cross-linking reaction

1. Protein sample:
   • protein concentration = 0.5–1.0 µg/µl in 50 mM HEPES pH 7.5-8.3, 150 mM NaCl;
   • starting material: Typically 30-50 µg protein can afford three MS runs in Q-Exactive with the NL signal at 1-2 E9.
   Note: (1) If you know the KD of the protein complex in question, aim for a concentration that is well above the KD value (e.g. 5x or 10x). If not, aim for 1.0 µg/µl.
   (2) Chemicals containing amino groups will compete with proteins in the cross-linking reaction, so Tris buffers must not be used. Phosphate buffers are OK.
   (3) Low concentrations of DTT (e.g. 1 mM) or detergent (e.g. 0.5% Triton X-100 or less, but keep as low as possible because detergent can greatly compromise MS analysis), 10 mM MgCl₂, and up to 1 M NaCl are OK. It also seems fine to have 20% glycerol, but we cannot say it with certainty for a lack of careful comparison.
   (4)The minimum volume of a protein sample is 10 µl.
   (5) t is highlyrecommend that freshlyeluted samples be cross-linked immediately if the buffer is compatible with the cross-linking reaction.
2. Dissolve Leiker in DMSO (keep DMSO in a desiccator, make fresh before each use or aliquot into tubessealed with parafilmand store in a desiccator at -20 °C). Adjustthe stock concentration with DMSO and make sure that the amount of DMSO in the cross-linking reaction does not exceed 10% by volume. 25 or 50 µg/µl stocks are usually prepared.
   • add the cross-linker solution to the protein sample at a 1:4 ratio (cross-linker-to-protein, w/w), mix well.
   Note: (1) The ratio above is based on the optimized cross-linking condition for a ten standard protein mixture using Leiker at pH 8.0, room temperature. It is recommended that several protein:cross-linker ratios be tested in the beginning, from 16:1, 8:1, 4:1, 2:1, 1:1 to 1:2 (w/w). After cross-linking, we want the intensity of individual subunits to reduce by 50–80% on SDS-PAGE (definitely no more than 90% to avoid over-cross-linking), and reasonably intense high Mw bands/smear by Coomassie stain.
3. Cross-link at room temperature for 0.5 h or 1 h.
4. Terminate the reactionby adding 1 M ammoniumbicarbonate to a final concentration of 20 mM and incubating at room temperature for 20 minutes.
5. Run 5 µg sample on SDS-PAGE and stain the gel with Coomassie Blue to examine the cross-linking reaction. The rest is for acetone precipitation, in-solution digestion, enrichment and MS analysis.
    

Acetone precipitation

1. Add 6x volume of acetone that is pre-cooled at -20 ºC.
2. Mix well and keep at -20 ºC for 30 min to overnight.
3. Spin in a refrigerated microfuge at top speed for 20 min.
4. Aspirate most of the supernatant and leave the last drop so as not to disturb the pellet.
5. If there is detergent in the sample, wash the pellet with 500 µl cold (-20 ºC) acetone twice.
    

Tryptic Digestion

1. Dissolve proteins in 20 µl 8 M urea, 100 mM Tris pH 8.5. Sonicate until a clearyellow solution is obtained.
2. Add 1 µl 100 mM TCEP (5 mM final concentration). Incubate at RT for 20min.
3. Add 0.4 µl 500 mM iodoacetamide (10 mM final concentration). Incubateat RT for 15 min in the dark.
4. Dilute sample by a factor of 4 with 100 mM Tris pH 8.5 (i.e. add 60 µl of Tris to 20 µl of sample, final volume = 80 µl, final concentration of urea = 2 M)
5. Add 100 mM CaCl₂ to a final concentration of 1 mM to enhance the activity of trypsin and increase its stability (i.e. 0.8 µl/80 µl).
6. Add methylamine to 20 mM to reduce carbamylation. (i.e. 1.33 µl 1.2 M CH3NH2 solution, 60x)
7. Add in 0.5 µg/µl trypsin at 50:1 ratio (w/w).
8. Incubate at 37 ºC for 16-18 h in the dark.

Enrichment on Streptavidin beads

Buffer:

Binding buffer: 20 mM HEPES pH 8.0, 150 mM NaCl 
Wash buffer 1: 20 mM HEPES pH 8.0, 1 M NaCl 
Wash buffer 2: 10% ACN in H₂O

Elution buffer: 300 mM Na₂S₂O₄ in 6 M urea/ 2 M thiourea, 10 mM HEPES pH8.2

Note: (1) to make 1 mL 6 M urea/ 2 M thiourea, mix 360.3 mg urea, 152.2 mg thiourea, 20 µl 1 M HEPES pH8.2 and 590 µl H₂O.
(2) to make 1 mL elution buffer, add 52.2 mg Na₂S₂O₄ to 1 mL 6 M urea/ 2 M thiourea.

(3) store Na₂S₂O₄ powder in a desiccator.

1. Bead Preparation (High Capacity Streptavidin Agarose Resin, Pierce): Transfer the appropriate amount of beads to a fresh 1.5 mL eppendorf tube. Wash the beads twice with 200 µl binding buffer. Centrifuge at 2500 ´g for 1-2 min.
   Note: (1) 20 µl slurry is enough for 50 µg starting protein.
   (2) to reduce the loss of beads, use a 1 mL syringe needle connected to a vacuum pump to remove the supernatant. Agitation of beads is avoided by sticking the needle to the wall of tubes.
2. Mix the tryptic digest with equal volume of binding buffer to reduce the concentration of urea. Add the sample to the washed beads and incubate at RT for 2 h with end-to-end rotation. The beads turn yellow upon binding of Leiker-linked peptides.
3. Centrifuge, save the flow-through in case of inefficient binding.
4. Wash the beads successively with wash buffer 1, H2O, wash buffer 2, and H2O. 1 mL buffer is applied every time and incubate for 5 min with rotation. Remove the final wash buffer as much as possible.
5. Add 5× bed volumes of elution buffer to the beads to release cross-linked peptides. Incubate at 37 °C for 30 min with rotation. The beads turn back to their original color after elution. Collect the sample with a narrow-end pipette tip.

Desalt

1. Acidify recovered peptides with 5% FA and load to a desalting column (250 µm × 2 cm, C18, 3 µm).
2. Wash the column with 0.1% FA over 10 column volumes (i.e. 10 µl).
3. Elute the sample with 10 µl 70% ACN/0.1%FA. Dry the solution by SpeedVac and reconstitute in 0.1% FA. Take one third of sample for one mass specanalysis.
   Note: Keep the flow rate at 1-1.5 µl/min at each step.

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