MBJ Selenate Mass Spec
Background
Sodium Selenate (Na2Se04) activates PP2A (1).
CTA and Selenate experiments show that male Sprague Dawley rats given systemic injections of 0.5mg/kg sodium selenate two hours before taste aversion conditioning form attenuated taste aversions that extinguish rapidly. Selenate also decreases LiCl-induced phospho-MAP kinase in the NTS and lateral PBN.
This experiment will use LCMS to identify differences in protein phosphorylation in the brain two hours after rats receive 0.5mg/kg selenate vs vehicle.
Agenda
07/15/2023 - Tissue Collection: 10 rats (5 males, 5 females) were overdosed with euthasol and rapidly decapitated two hours after receiving 1ml/kg i.p. injections of 0.5mg/kg sodium selenate or vehicle (0.15M NaCl). Brains were dissected and washed in ice-cold PP2A storage buffer (50mM Tris-HCl, pH 7.4, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 250mM Sucrose, 0.1%(v/v) N-PER detergent, 1 Pierce protease inhibitor mini tablet per 10mL solution), and frozen at -80*C.
| Subject | Sex | Treatment | Weight (g) | Injection vol (ml) | Injection time (PM) | Time of brain dissection (PM) |
|---|---|---|---|---|---|---|
| MBJ01 | M | NaCl | 371 | 0.37 | 2:17 | 4:28 |
| MBJ02 | F | Sel | 257 | 0.26 | 2:26 | 4:34 |
| MBJ03 | M | Sel | 348 | 0.35 | 2:33 | |
| MBJ04 | F | NaCl | 250 | 0.25 | 2:40 | |
| MBJ05 | M | NaCl | 346 | 0.35 | 2:46 | 5:00 |
| MBJ06 | F | Sel | 247 | 0.25 | 2:53 | |
| MBJ07 | M | Sel | 324 | 0.33 | 2:59 | |
| MBJ08 | F | NaCl | 245 | 0.25 | 3:04 | |
| MBJ09 | M | NaCl | 335 | 0.34 | 3:10 | |
| MBJ10 | F | Sel | 263 | 0.26 | 3:16 | 5:33 |
7/28/2023 - Protein Extraction: For each frozen tissue sample (MBJ01-10), Amygdalar lobe (A) and caudal brainstem (B) regions were dissected and homogenized in PP2A storage buffer (50mM Tris-HCl, pH 7.4, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 250mM Sucrose, 0.1%(v/v) N-PER detergent, 1 Pierce protease inhibitor mini tablet per 10mL solution)
- 1:3 ratio of brain tissue:buffer
- Dounce homogenization: 50-60 strokes on ice
- The homogenate was centrifuged at 100,000 x g, 4°C for 1 hour
- The supernatants were collected and stored at 4°C overnight
7/29/2023 - Sample clean-up: detergent and salt removal The supernatants were buffer exchanged into TEE buffer (50mM Tris-HCl pH 7.4, 0.1mM EDTA, 0.1mM EGTA) using PD-10 desalting columns (Cytiva #17085101) The samples were then aliquoted and frozen at -20°C
7/31/2023 - Bradford and BCA assays to determine protein concentration Both Bradford and BCA Assays were conducted to determine the protein concentration. Results generated from both assays were nearly identical +/- 0.02ug/ml The protein concentrations estimated from the BCA assay were used for all subsequent calculations
9/18/2023 - Reduction/Alkylation/Digestion
Reduction
- Add 60 uL of each sample to a premade tube of 0.04g urea → [final] = 8M urea
- Vortex until dissolved
- Place in 37 deg C water bath for 2 mins
- Add 5 uL TCEP bond breaker to each sample
- pipette/mix 5x
- Pulse 5 sec
- Incubate in 37 deg C water bath for 60 mins
Alkylation
- Add 10 uL of 20mM iodoacetamide stock solution to each sample
- Vortex 2 seconds
- Pulse 5 seconds
- Incubate in 37 deg C water bath for 20 mins
- Add 400 uL water to each sample → ~500 uL final volume
Digestion
- Reconstitute 20ug Trypsin (Promega #V5111) by adding 200 ul of supplied buffer → [final] = 1ug/10ul trypsin
- Add enough trypsin for a 1:20 ratio of trypsin:protein
- Incubate all samples in 37°C water bath overnight
Next day:
- Remove samples from water bath
- Quench with 2.6 uL of 10% Formic Acid
- Store at -20°C
Final sample
- Total number of samples = 17
- 09A, 09B, and 10B were excluded from this batch due to possible cross-contamination
- Buffer (before reduction/alkylation/digestion) = 50mM Tris-HCl pH 7.4, 0.1mM EDTA, 0.1mM EGTA
- Final volume = ~500uL per sample
09/21/2023 - Samples delivered to Translational Lab for Mass Spec analysis
- Samples passed through S-trap column for final clean-up (removal of urea and salts)
References
Corcoran NM, Martin D, Hutter-Paier B, Windisch M, Nguyen T, Nheu L, Sundstrom LE, Costello AJ, Hovens CM. Sodium selenate specifically activates PP2A phosphatase, dephosphorylates tau and reverses memory deficits in an Alzheimer’s disease model. Journal of Clinical Neuroscience 17: 1025- 1033, 2010.[1]
People
Marena Bass