Protein Extraction SOP

proteomics
Author

hxyv

Published

July 21, 2023

Depletion of Albumin and IgG from Plasma Samples

(High-Select™ Top14 Abundant Protein Depletion Resin, Thermo [A36370])

  1. Pool plasma samples from the same group into one (8 \(\mu L\) × 5 = 40 \(\mu L\)).

  2. Equilibrate the depletion spin column to room temperature.

  3. Remove the column screw cap and add up to 5 \(\mu L\) of sample directly to the resin slurry in the column.

  4. Cap the column and invert the column several times until the resin is completely homogeneous in solution.

  5. Incubate the mixture in the column with gentle end-over-end mixing for 30 min at room temperature. Make sure the sample mixes with the resin during incubation period. Alternatively, gently vortex every few minutes.

  6. After incubation, snap off the bottom closure and lossen the top cap. Place the mini column into a 1.5 \(mL\) collection and centrifuge at 1,000\(\times g\) for 2 min, after centrifuge, the liquid in the collection tube is about 300 \(\mu L\).

  7. Discard the column containing the resin, store the collection tube at 4\(^\circ C\) for future experiments.

BCA Assay

(BCA Protein Assay Kit, Sigma [71285-3])

  • Reagent A: contains bicinchoninic acid (BCA), sodium carbonate, sodium tartrate and sodium bicarbonate in 0.1M NaOH.
  • Reagent B: contains 4% (\(w/v\)) copper (II) sulfate pentahydrate.
  • Protein standard (BSA) solution: the stock BSA concentration is 1 \(mg/mL\) in 0.15M NaCl with 0.05% sodium azide as a preservation.

Prepare BCA Working Regent (WR)

The BCA working reagent is prepared by mixing 50 parts of Reagent A with 1 part of Reagent B. Mix the BCA working regent until it is light green in color (Table 1). The BCA working reagent (Reagent A mixed with Reagent B) is stable for one day.



Table 1: Volume of BCA Working Reagent to prepare.
Number of wells in a 96 well plate assay Reagent A (\(mL\)) Reagent B (\(mL\)) Total volume of BCA working reagent (\(mL\))
30 7 0.14 7.14
40 8 0.16 8.16
80 16 0.32 16.32
96 19 0.38 19.38
127 25 0.5 25.5

Prepare BSA standard solution

The BSA stock solution is 2 \(mg/mL\). For the dilution steps of BSA series standard solutions, refer to Table 2 below and dilute with water (200, 400, 600, 800, 1,000 \(\mu g/mL\))



Table 2: Example of standard assay setup.
No. Volume of PBS (\(\mu L\)) Volume of stock BSA 2 \(mg/mL\) (\(\mu L\)) Final conc. (\(\mu g/mL\)) Total volume (\(\mu L\))
A 50 50 1,000 100
B 180 120 800 300
C 25 75 \(\mu L\) of B 600 100
D 70 70 \(\mu L\) of B 400 140
E 50 50 \(\mu L\) of D 200 100
F 100 0 0 100



Dilution of sample solution

Take 45 \(\mu L\) of samples in step 1.6, and dilute with 45 \(\mu L\) PBS.

Measure

  1. Pipette BSA series standard solution, blank, sample solution to a 96-well plate, 25 \(\mu L\) per well, prepare three replicates, add 200 \(\mu L\) working reagent to each well, gently mix for 30 s, and put on the cover.

  2. Incubate the samples for:

    1. 60 \(^\circ C\) for 15 minutes Or
    2. 37 \(^\circ C\) for 30 minutes Or
    3. 25 \(^\circ C\) (Room Temperature) from 2 hours to overnight

  3. If required, allow the plates to cool to room temperature.

  4. Measure the absorbance of the solution at 562 \(nm\). Create an assay table as needed and a standard curve based on either the BSA protein standard concentration or on the amount of protein present in the BSA protein standard.

  5. Determine protein concentration by comparison of the absorbance of the unknown samples to the standard curve prepared using the BSA protein standards.

Proteolysis (100 \(\mu g\) total protein)

Sample vacuum concentration (To change to urea solution)

  1. Transfer about 100 \(\mu g\) of the sample in step 1.6 into a 1.5 \(mL\) centrifuge tube, use a vacuum concentrator (SpeedVac) at room temperature for 1-2 hours, gently mix samples at 1 hour.

  2. Dissolve with 50 \(\mu L\) of 8M Urea (weigh 14.42 \(g\) of Urea and dissolve it in 1 \(L\) of ultrapure water).

DTT reduction

Add 2.63 \(\mu L\) 100mM DTT (prepared with 10mM Ammonium Bicarbonate solution), incubate at 56\(^\circ C\) for 30 min, the final concentration of DTT should be 5mM.

  • 10mM Ammonium Bicarbonate (ABC) solution: weigh 31.63 \(mg\) ABC and dissolve in 40 \(mL\) ultrapure water.
  • 100mM DTT (154.25): weigh 15.425 \(mg\) DTT and dissolve in 1 \(mL\) 10mM ABC.

Alkylation with iodoacetamide

Cool to room temperature, add 2.77 \(\mu L\) 220mM IAM, and incubate for 30 min at room temperature in the dark, the final concentration of IAM should be 11mM.

  • 25mM Ammonium Bicarbonate (ABC) solution: weigh 79 \(mg\) ABC and dissolve in 40 \(mL\) ultrapure water.

  • 220mM IAM (184.96): weigh 20.4 \(mg\) IAM and dissolve in 500 \(\mu L\) ABC (IAM should be stored in a dark environment).

Trypsinization

  1. Add seven volumes of 25mM ABC to the samples in step 3.3 (387.8 \(\mu L\) in this case), and control the final concentration of Urea less than 1M.

  2. Add 2 \(\mu L\) of 1 mg/mL Promega trypsin [V5111] (protein:enzyme mass ratio = 50:1), and overnight digestion for 12-16 hours.

  3. Add 4.5 \(\mu L\) 10% TFA to stop digestion, the final concentration of TFA is 1%, incubate on ice for 10 min, centrifuge at 5,000\(\times g\) 2 min, and carefully collect the supernatant.

Sample Desalting

(MonoSpin C18 spin column S 100 pcs, GL Science [5010-21701])

Desalination and concentration

  1. Column balance: place the spin column on the waste collection tube, add 200 \(\mu L\) acetonitrile, centrifuge at 5,000\(\times g\) 1 min at room temperature, and discard the waste in the collection tube.

  2. Add 200 \(\mu L\) 0.1% \(TFA-H_2O\), centrifuge at 5,000\(\times g\) 1 min at room temperature, and discard the waste in the collection tube.

  • 0.1% TFA: mix 1 \(\mu L\) TFA with 999 \(\mu L\) \(H_2O\).

Sample loading

  1. Transfer the sample in step 3.4.3 to the spin column, centrifuge at 5,000\(\times g\) 2 min, and discard the waste in the collection tube.

  2. Add 300 \(\mu L\) 0.1% \(TFA-H_2O\), centrifuge at 5,000\(\times g\) 1 min, and discard the waste collection tube.

Elution (twice)

Replace the sample collection tube, add 200 \(\mu L\) 60% acetonitrile solution, and centrifuge at 5,000\(\times g\) 1 min.

  • 60% acetonitrile: mix 480 \(\mu L\) acetonitrile with 320 \(\mu L\) \(H_2O\).

Dry and concentration

Dry the sample in a vacuum concentrator (SpeedVac) at 30\(^\circ C\) for 1-2 hours (to remove acetonitrile), gently mix samples at 1 hour.

TMT labeling

(TMTsixplex™, Thermo [90066])

  1. Resuspend samples in step 4.4 with 100 \(\mu L\) 50mM TEAB.

  2. Immediately before use, equilibrate the TMT Label Reagents to room temperature, and avoid light. For the 0.8mg vials, add 41μL of anhydrous acetonitrile to each tube. Allow the reagent to dissolve for 5 minutes with occasional vortexing. Briefly centrifuge the tube to gather the solution.

Note: Reagents dissolved in anhydrous acetonitrile are stable for one week when stored at -20°C. Anhydrous ethanol can be used as an alternative solvent to dissolve reagents but is not recommended for stock solution storage.

  1. Carefully add 41 \(\mu L\) of the TMT Label Reagent to each 100 \(\mu L\) sample (25-100 \(\mu g\) protein digest), and avoid light.

Note: Labeling more than 100 \(\mu g\) of protein digest per reaction requires an additional TMT label reagent.

  1. Incubate the reaction for 1 hour at room temperature, and avoid light.

  2. Add 8 \(\mu L\) of 5% hydroxylamine to the sample and incubate for 15 min to quench the reaction, and avoid light.

  3. Combine samples in equal amounts in a new microcentrifuge tube and store at -80\(^\circ C\).

  • 50mM TEAB: mix 25 \(\mu L\) TEAB with 475 \(\mu L\) \(H_2O\).
  • 100mM TEAB: mix 2 \(\mu L\) TEAB with 18 \(\mu L\) \(H_2O\).
  • 5% Hydroxylamine: mix 2 \(\mu L\) 50% hydroxylamine with 18 \(\mu L\) 100mM TEAB.