In-solution digestion protocol

In order to prepare samples for mass spectrometry measurements, proteins need to be converted into peptides and sample must be purified from any other contaminating content like detergents, salts, nucleic acids etc.

1 Crucial steps

1.1 Denaturation

Protein denaturation is a process in which the application of external stress leads proteins to lose all the structures present in their native state. However, denaturation reactions are not strong enough to break the peptide bonds. Therefore, they don’t lose their primary structure (Figure 1). This stress promotes the disruption of protein interactions and can be applied as a chemical stress, a physical stress, or both.

1.2 Reduction and Alkylation

A protein sample is typically reduced & alkylated to break disulfide bridges and ‘cap’ the reduced cysteines to prevent re-oxidation.

1.3 Digestion

Protein digestion is performed by enzyme called endopeptidase or endoproteinase. These enzymes break peptide bonds of nonterminal amino acids (i.e. within the molecule). In case of proteomics in mass spectrometry, the enzyme most commonly used is called trypsin, and it cuts after Arg (R) or Lys (K).

2 Solvents

  • SLS 4% (in 50mM TEAB)

  • DTT = 400mM (in H2O)

  • IAA = 550 mM (in H2O)

  • Lys-C = 0,5 µg/µl

  • Trypsin = 0,2 µg/µl

  • TFA 10%

  • TEAB buffer 50 mM

  • reconstitution buffer 0.1% TFA / 2% ACN (in H2O)

3 Sample preparation

  1. You will be given an aliquot of a cell pellet lysate in 4% SLS1

  2. Estimate protein concentration via BCA Protein Assay

    1. Prepare BCA working reagent (WR) by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B) to achieve 150 µl of the reagent per sample.

    2. Pipette 5 µl of each albumin (BSA) standard and unknown sample into a microplate well (working range = 20–2000 µg/mL).

    3. Add 150 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds (you can also use ThermoMixer, hold the plate in hands, do not exceed 700rpm).

    4. Cover plate and incubate at 37°C for 30 minutes.

    5. Measure the absorbance at or near 562 nm on a plate reader.

  3. Based on the BCA measurements, take 30 µg of protein for next steps

  4. Add DTT stock (40x - 1µl of DTT to 40µl of the lysate) to get the final concentration of 10 mM. Incubate in ThermoMixer with vortex for 10 min at 90°C

  5. Add IAA stock (40x) to get the final concentration of 55 mM. Vortex and incubate in the dark for 30 min at RT (22°C)

  6. Take a small aliquot containing 5 µg of proteins for a control on the NuPage-gel

  7. Dilute the reduced and alkylated lysate 4x with 50 mM TEAB buffer to a final SLS concentration of 0.5% SLS2

  8. Add Trypsin (stock = 0,2 µg/µl) at an ratio of 1:50 (enzyme:substrate) and incubate at 37°C overnight (alternatively 2h at 40°C)

  9. Spin down the sample

  10. Take a small aliquot containing 5 µg of peptides for a control on the NuPage-gel

  11. Stop the digestion by acidifying with 10% TFA to a final concentration of 1.5% TFA (pH must be <2). Use more acid if needed! SLC will precipitate

  12. Let samples rest for 10 min at room temperature

  13. Centrifuge the samples at full speed for 10 min, 4°C3

  14. Desalt the supernatant via SepPak C18 columns (go to appropriate protocol)

Footnotes

  1. Cell pellets from Yeast, E.coli and Hela underwent an incubation at 90°C for 10 min with shaking and a 2 minutes sonication, in order to denature proteins and destroy all the DNA.↩︎

  2. Trypsin is a protein as well, and it can also be denatured in high SLS concentration. In order to preserve enzyme activity, we have to dilute the detergent.↩︎

  3. If the pellet doesn’t really stick to the bottom of the Eppi, try to lower the temperature of the samples to near freezing point. Helps with precipitation.↩︎