Skip to Main Content U.S. Department of Energy
Biological Banner

Protocols

Soluble Protein Sample Preparation - Prokaryotic Organisms

Contents

Purpose

This procedure is to be used for preparation of peptide samples from soluble proteins from prokaryotic organisms. The peptide samples will be used for mass spectrometry based proteomic analysis.

Responsibility

All personnel performing this experiment will be responsible for reading, understanding and complying with the provisions outlined within this method.

It is the responsibility of the appropriate manager to ensure that this document is reviewed and updated as needed.

Safety

Before beginning any of the procedures involved in this method, each individual must read and sign the Chemical Hygiene Plan developed for PNNL. Material Safety Data Sheets (MSDS) for the chemicals and reagents are available online.

Interferences

Ensure that when possible, low-retention microcentrifuge tubes are used throughout the procedure to reduce polymer leaching effects. Ensure that samples stay on ice during the procedure unless otherwise noted.

Ensure that original sample is free from detergents. If the sample contains a small amount of media salts or other chemicals, these may interfere with the protein assay. Consult the instruction manual for the protein assay to ensure that this will not occur.

Caution

Ensure that all training based in the Integrated Operations System (IOPS) has been completed and all safety procedures are being followed.

Apparatus

Items needed for sample preparation are:

  • Vortexer mixer
  • 0.1 mM Zirconia/Silica Beads
  • Bead beater
  • Centrifuge at 4ºC
  • Ultracentrifuge (with appropriate rotors and tubes)
  • Low-retention microcentrifuge tubes (0.6, 1.5 & 2.0 mL)
  • 60ºC incubator
  • 37ºC incubator
  • 15- and 50-mL Falcon tubes
  • Cryovials
  • 26-gauge syringe needle

Reagents

Prepare all reagents in appropriate containers, preserving sterility when necessary. Ensure that the water used to prepare solutions is of Nanopure or Milli-Q quality (~18 megohm•cm or better).

  1. 100 mM NH4HCO3, pH ~8 buffer
  2. BCA or Coomassie Protein Assay Reagents
  3. Urea (in solid form)
  4. Thiourea (in solid form)
  5. 50 mM Dithiothreitol (DTT)
  6. 1 M CaCl2
  7. Sequencing-grade modified Trypsin

Procedure

  1. Pelletize cells and resuspend in 100 mM NH4HCO3, pH ~8 buffer (wash cells once if necessary with buffer)
  2. Transfer reconstituted cells to low-retention 0.6-mL microcentrifuge tubes (make sure no more than 200 μL is in each tube)
  3. Add 0.1 mM Zirconia/Silica Beads to the 0.6 mL mark on each tube (make sure there is a layer of dry beads on top of cells)
  4. Bead beat at max speed for 3 minutes, after removing immediately placing on ice.
  5. Poke hole in base of 0.6-mL microcentrifuge tube with 26-Ga needle, nest inside an empty and decapped 1.5-mL microcentrifuge tube, and centrifuge for 5 min at 14,000 rpm, 4ºC.
  6. If a rinse of the beads is desired, add 100-200 µL of bufer to the top of the beads in the 0.6-mL tube. Place 0.6-mL tube in a second 1.5-mL low-retention microcentrifuge tube, and centrifuge again for 5 min at 14,000 rpm, 4ºC.
  7. Resuspend lysate(s) and combine into one tube for each sample (if necessary).
  8. Centrifuge lysate at 4,000 rpm, 4ºC for 2 minutes to spin out any whole cells.
  9. Transfer supernatant to ultracentrifuge tubes, balance appropriately, and ultracentrifuge at 100,000 rpm for 10 minutes @ 4ºC. Draw off supernatant from this spin and use for soluble protein preparation (the pellet is used for the insoluble protein preparations – see appropriate SOP). If desired, the membrane pellet can be resuspended (using either vortexing and pipetting or sonication) in either water or buffer and centrifuged as above again to “wash” pellet. Transfer the supernatant from the second spin into the vial containing the soluble protein from the first spin.
  10. Perform Coomassie or BCA Protein Assay on soluble portion.
  11. Determine volume of sample with pipet and calculate mass of sample in µg.
  12. Add mini Proteome as per mini Proteome calculator
  13. Add powdered form of Urea to sample to a target concentration of 7 M (424 mg/mL solution).
  14. Add powdered form of Thiourea to sample to a target concentration of 2 M (152 mg/mL solution).
  15. Prepare a 50 mM solution of Dithiothreitol (DTT – 7.7 mg in 1 mL nanopure water). Add an appropriate volume of DTT solution to obtain a 5 mM concentration in the sample.
  16. Incubate the sample at 60ºC for 30 min.
  17. After removing the sample from the incubator, add buffer to the trypsin vial(s) to obtain desired concentration (i.e. add 20 uL to vial to obtain 1 ug/uL trypsin solution), and pre-activate trypsin by incubating vial(s) for 5-10 minutes at 37ºC.
  18. Dilute the sample 10-fold with 100 mM NH4HCO3 to reduce the salt concentration.
  19. Add sufficient amount of a 1M solution of CaCl2 to obtain a sample concentration of 1 mM CaCl2.
  20. Digest sample for 3 hours with Trypsin @ 37ºC at a concentration of 1 unit trypsin/50 units protein.
  21. After trypsin incubation, immediately place sample on ice (or quick freeze until needed to finish sample preparation).
  22. Perform C18 SPE clean-up following the appropriate standard protocol.

BER-PNNL Proteomics

References

Contacts

Content

WebMaster