Apr 25, 2024
PURIFICATION OF PROTEINS FROM PFA FIXED SAMPLES BAK_WITH BIOTIN PULLDOWN FOR LC-MS/MS_2023
- 1Rush University
Protocol Citation: Bryan Killinger 2024. PURIFICATION OF PROTEINS FROM PFA FIXED SAMPLES BAK_WITH BIOTIN PULLDOWN FOR LC-MS/MS_2023. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3xyylk5/v1
Manuscript citation:
Killinger BA, Mercado G, Choi S, Tittle T, Chu Y, Brundin P, Kordower JH. Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis. NPJ Parkinsons Dis. 2023 Mar 25;9(1):43. doi: 10.1038/s41531-023-00491-3. PMID: 36966145; PMCID: PMC10039879.
Choi SG, Tittle T, Garcia-Prada D, Kordower JH, Melki R, Killinger BA. Alpha-synuclein aggregates are phosphatase resistant. bioRxiv [Preprint]. 2024 Apr 9:2023.11.20.567854. doi: 10.1101/2023.11.20.567854. PMID: 38645137; PMCID: PMC11030248.
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
We use this protocol and it's working
Created: February 27, 2024
Last Modified: April 25, 2024
Protocol Integer ID: 95966
Funders Acknowledgement:
NINDS
Grant ID: 1R01NS128467
Michael J. Fox Foundation
Grant ID: ASAP-024442
Abstract
This protocol details the purification of proteins from PFA fixed samples and extraction of proteins from formalin-fixed tissues. Also included, biotin pulldown prior to LC-MS/MS. Samples generated for this protocol have been used for mass spectrometry, immunoblotting, and pulldowns.
Attachments
Image Attribution
Killinger BA, Mercado G, Choi S, Tittle T, Chu Y, Brundin P, Kordower JH. Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis. NPJ Parkinsons Dis. 2023 Mar 25;9(1):43. doi: 10.1038/s41531-023-00491-3. PMID: 36966145; PMCID: PMC10039879.
Materials
Wash Buffer (1L):
| A | B | |
| 10X TBS pH 7.6 | 100 mL | |
| Triton X-100 | 10 mL | |
| SDS | 1 g | |
| 0.5M EDTA pH 8.0 | 2 mL |
Dissolve in milliq water. Up to final volume 1L. Store at room temperature for 6 months.
Reversal Buffer (RB) (500ML):
| A | B | |
| SDS | 25g | |
| 0.5M EDTA pH 8.0 | 2 mL | |
| Tris-Base | 30.35 g | |
| NaCl | 4.38 g |
Dissolve in milliq water. pH to 7.6. Up to final volume 500mL. Store at room temperature for up to 1 year.
PMSF solution (100 mM). Acute toxicity, handle powder carefully.
1. Place 1.5mL tube onto scale, tare.
2. Add small amount of PMSF to tube, record weight.
3. Add calculated volume of isopropanol to PMSF. Mix well. To calculate: PMSF mass (mg) / 17.4 = volume of isopropanol.
Stable in isopropanol at RT for at least 6 months. Stable for ~15 min once added to aqueous solution.
Fixation solution:
| A | B | |
| Ethanol | 50% | |
| Acetic acid | 10% |
Procedure Day 1 (Extract proteins and test concentration)
Procedure Day 1 (Extract proteins and test concentration)
1h 35m
Wash sections in DM 3 X 10 min.
Wash sections in DM for 00:10:00 (1/3)
10m
Wash sections in DM for 00:10:00 (2/3)
10m
Wash sections in DM for 00:10:00 (3/3)
10m
Place sections in 1.5mL Eppendorf tube.
Add 0.5 mL of reversal buffer.
Briefly sonicate on low power to disperse tissue.
Add 5 µL of 100 millimolar (mM) PMSF. Mix well. Quick spin.
Heat on block for 98 °C for 00:30:00 with cap locks.
30m
Remove from heat block carefully (caps will pop if not careful).
After 00:05:00 of cooling, vortex well.
5m
Centrifuge at 22000 x g for 00:30:00 Room temperature .
30m
Collect S1 (extracted proteins).
Western blot (Optional)
Western blot (Optional)
Perform methanol/chloroform cleanup on 100 µL of S1.
Resuspend resulting pellet in 100 µL 5% SDS.
Perform BCA assay on 100 µL . Each BCA test takes 20 µL . The remaining volume can be used for western blot. Alternatively, more protein can be prepared from S1 if needed.
Calculate volume required for 20 µg protein.
20ug / concentration of S1 ug/ul = ul of sample required
Procedure Day 2 (Capture biotinylated proteins and wash)
Procedure Day 2 (Capture biotinylated proteins and wash)
10h 3m
Add S1 to 10 mL TBST in 15mL conical tube. Mix well.
Add 40 µL of prepared magnetic streptavidin beads to each tube.
Nutation for 01:00:00 Room temperature .
1h
Place tube on magnetic stand for 00:01:00 (until all beads have been drawn out of solution).
1m
Carefully remove supernatant no to disturb beads. Add 10 mL wash buffer.
Nutation for 00:30:00 .
30m
Place tube on magnetic stand for 00:01:00 (until all beads have been drawn out of solution).
1m
Add 10 mL wash buffer.
Nutation for 00:30:00 .
30m
Place tube on magnetic stand for 00:01:00 (until all beads have been drawn out of solution).
1m
Add 10 mL wash buffer
Nutation Overnight at 4 °C .
8h
Day 3 (Elute captured proteins)
Day 3 (Elute captured proteins)
12m
Place tube on magnetic stand for 00:01:00 (until all beads have been drawn out of solution).
1m
Carefully remove supernatant no to disturb beads.
Add 1 mL of wash buffer.
Mix samples until beads are suspended in wash buffer.
Using a pipette with tip cut off, transfer suspended beads to a 1.5mL protein low bind tube.
Place tube on magnetic stand for 00:01:00 (until all beads have been drawn out of solution).
1m
Remove buffer and surface wash with milliq 2X.
Quick spin and remove liquid from tube (should only have beads left).
Add 80 µL 1X SDS-page sample buffer containing reducing agent.
Mix well and quick spin.
Place on heat block set to 98 °C for 00:10:00 .
10m
Mix well and quick spin.
Place on magnet.
Transfer eluent to two separate lo-bind tube and discard used beads. 35 µL in 1 tube (used for premass spec QC), the remaining 45 µL in another (used for mass spec).
Immediately place in -80 °C for long-term storage.
Day 4 (Prepare proteins for Mass Spec)
Day 4 (Prepare proteins for Mass Spec)
3h 8m
Prepare Bis-Tris 4-12% wedge gel 15 well.
Make 800 mL 1X MOPS running buffer.
Load 5 µL of MW standard in one side of gel.
For MS load exactly 40 µL of the samples into wells. Careful not to spill into opposing wells. Only load samples into every other well to prevent cross contamination.
Ensure all wells have a buffer. For empty wells fill with 40 µL 1X loading buffer.
Run the gel at 150V for a few minutes. Watch carefully until the sample has completely entered gel.
Stop running, remove gel with clean gloves, and clean equipment.
Fix gel in 100mL fixation solution (refer materials section) for 01:00:00 Room temperature .
1h
Wash gel in several changes of milliq, until gel has swollen to original size.
Place in 100 mL colloidal Coomassie blue stain solution. Cover loosely, and heat in microwave for few minutes until solution just starts to boil.
Incubate gel in heated colloidal Coomassie blue stain solution for 00:08:00 .
8m
Remove gel from stain solution and wash several times with milliq water. Clear background is achieved with ~02:00:00 of washing.
2h
Using a clean razor excise the entire sample from the gel.
Place each sample into a clean 1.5mL tube. Add 500 µL of milliq water.
Samples can be stored at 4 °C until submitted to mass spec core.
Optional: Pre mass spec QC
Optional: Pre mass spec QC
45m
Estimating capture concentration - Purpose: To provide mass spec core with a rough estimate of the amount of protein in the capture sample.
Note
This prevents potential overloading, or digestion problems in the mass spec core.
Prepare gel as described above (Day 4, Steps 43-45)
Load 20 µL of QC aliquot into wells.
Load 2 µg BSA into 1 well.
Run the gel at 150V for approximately 00:45:00 or until the die has reached end of gel.
45m
Stain gel with Coomassie as described above (Day 4, Steps 50-54)
Image on Odyssey using NIR.
Forward image to NW mass spectrometry core.
Example of results; order = 2ug BSA, 3 captures, 2ug BSA, 3 captures, 2ug BSA, 3 captures
Dot blot to estimate target enrichment
Dot blot to estimate target enrichment
8h 0m 30s
Cut PVDF membrane into ~1X3 inch piece.
Activate PVDF in 100% methanol for 00:00:30
30s
Equilibrate activated PVDF in diH20.
Just prior to blotting samples, place hydrated PVDF on wypall and quickly pat dry.
Place PVDF on new dry wypall.
Spot 1 µL of each sample onto PVDF.
Allow PVDF to dry completely. (~1h or Overnight )
8h
Reactivate spotted PVDF as described above and equilibrate in dH20.
Blot can now be probed with antibodies using standard protocols.