Facility Recommended:  Surfactant Enhanced In Gel Digestion of Proteins

 [4hr protocol suitable for samples >20ng]

Buffers and Solutions

Gel fragment preparation

  • Excise protein bands. Cut each into 1 mm pieces. Place into a low-binding tube (LoBind, Eppendorf). Also cut out a gel piece from a protein-free region as control.
  • Wash gel pieces with >10 volumes of Millipore water [~200µl] for 30 seconds, to wash out acetic acid.

Destaining

  • For Coomassie Blue staining, destain two times for 5 minutes or until colorless with 200µl 100mM (NH4)HCO3 / 50% Methanol (discard supernatants). Dehydrate for 2 minutes with 200µl 25 mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. Remove the solutions and discard. The gel pieces shrink and become white.
  • For Non-Destructive Silver staining, destain twice for 2 minutes or until colorless with 200µl of freshly prepared 1:1 solution of 100mM Sodium Thiosulfate [Na2S2O3] and 30mM Potassium Ferricyanide [K3Fe(CN)6]. Stop the reaction and wash out silver ions twice for 2 minutes with 500µl of Millipore water. Dehydrate for 2 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  The gel pieces shrink and become white.
  • For Sypro Ruby staining, no destaining necessary, Dehydrate for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  Remove the solutions and discard. The gel pieces shrink and become white.

     

  • Dry gel particles for 2 minutes in a vacuum centrifuge.

Reduction and Alkylation

  • Rehydrate gel pieces in 50µl of freshly prepared 25mM Dithiothreitol (in 25mM (NH4)HCO3). Reduce the proteins for 20 minutes at 56°C.
  • Cool the gel to room temperature, pipet off any residual liquid and add 50µl of freshly prepared 55mM Iodoacetamide (in 25mM (NH4)HCO3). Alkylate the proteins for 20 minutes at room temperature in the dark.
  • Pipet off the liquid and wash gel pieces with > 20 volumes of Millipore water [~400µl] for 30 seconds to remove any residual Iodoacetamide. Dehydrate gel pieces for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.
  • Dry gel particles for 2 minutes in a vacuum centrifuge.

Trypsin Digestion

  • Rehydrate gel pieces for 5 minutes at room temperature in 20µl digestion solution containing 200ng Trypsin (Promega Sequence Grade Modified) in 25mM (NH4)HCO3 / 0.01% ProteasMAXTM (Trypsin enhancing and extraction promoting surfactant from Promega Corporation, Madison, WI) [ pH =~8.5].
  • Overlay the rehydrated gel particles with a minimum amount of 25mM (NH4)HCO3 / 0.01% ProteasMAXTM to keep them immersed throughout the digestion. Incubate 2-3 hours at 42ºC.

Peptide Recovery and Solid-Phase Extraction

  • Flash-spin the digest and transfer solution to a new low-binding tube (LoBind, Eppendorf). Inactivate the trypsin and cleave surfactant with TFA [0.3% final concentration].
  • Centrifuge for 10 minutes at max speed to pellet cleaved surfactant, transfer digestion solution to a new tube.
  • Sample is ready for LC-based analysis.
  • Perform ZipTip-C18 column (Millipore) clean-up before spotting onto MALDI plate.

 

References

  • Saveliev, S. et al Promega Notes 99. 2008; 3–7

 

Facility Recommended: 

Surfactant Enhanced In Gel Digestion for Scarce Samples

 [4hr protocol suitable for samples <20ng]

Buffers and Solutions

Gel fragment preparation

  • Excise protein bands. Cut each into 1 mm pieces. Place into a low-binding tube (LoBind, Eppendorf). Also cut out a gel piece from a protein-free region as control.
  • Wash gel pieces with >10 volumes of Millipore water [~200µl] for 30 seconds, to wash out acetic acid.

Destaining

  • For Coomassie Blue staining, destain two times for 5 minutes or until colorless with 200µl 100mM (NH4)HCO3 / 50% Methanol (discard supernatants). Dehydrate for 2 minutes with 200µl 25 mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. Remove the solutions and discard. The gel pieces shrink and become white.
  • For Non-Destructive Silver staining, destain twice for 2 minutes or until colorless with 200µl of freshly prepared 1:1 solution of 100mM Sodium Thiosulfate [Na2S2O3] and 30mM Potassium Ferricyanide [K3Fe(CN)6]. Stop the reaction and wash out silver ions twice for 2 minutes with 500µl of Millipore water. Dehydrate for 2 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  The gel pieces shrink and become white.
  • For Sypro Ruby staining, no destaining necessary, Dehydrate for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  Remove the solutions and discard. The gel pieces shrink and become white.

     

  • Dry gel particles for 2 minutes in a vacuum centrifuge.

Reduction and Alkylation

  • Rehydrate gel pieces in 50µl of freshly prepared 25mM Dithiothreitol (in 25mM (NH4)HCO3). Reduce the proteins for 20 minutes at 56°C.
  • Cool the gel to room temperature, pipet off any residual liquid and add 50µl of freshly prepared 55mM Iodoacetamide (in 25mM (NH4)HCO3). Alkylate the proteins for 20 minutes at room temperature in the dark.
  • Pipet off the liquid and wash gel pieces with > 20 volumes of Millipore water [~400µl] for 30 seconds to remove any residual Iodoacetamide. Dehydrate gel pieces for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.
  • Dry gel particles for 2 minutes in a vacuum centrifuge.

Trypsin Digestion

  • Rehydrate gel pieces for 5 minutes at room temperature in 20µl digestion solution containing 50ng Trypsin (Promega Sequence Grade Modified) in 25mM (NH4)HCO3 / 0.01% ProteasMAXTM (Trypsin enhancing and extraction promoting surfactant from Promega Corporation, Madison, WI) [ pH =~8.5].
  • Overlay the rehydrated gel particles with a minimum amount of 25mM (NH4)HCO3 / 0.01% ProteasMAXTM to keep them immersed throughout the digestion. Incubate 2-3 hours at 42ºC.

Peptide Recovery and Solid-Phase Extraction

  • Flash-spin the digest and transfer solution to a new low-binding tube (LoBind, Eppendorf).
  • Additional extraction step: add 10-25µl of 2.5% TFA [depends on the gel size, enough to completely immerse gel pieces], vortex for 5 minutes, flash-spin and transfer liquid to previously collected aliquot.
  • Centrifuge for 10 minutes at max speed to pellet cleaved surfactant, transfer digestion solution to a new tube.
  • Sample is ready for LC-based analysis.
  • Perform ZipTip-C18 column (Millipore) clean-up before spotting onto MALDI plate.

 

    References

    • Saveliev, S. et al Promega Notes 99. 2008; 3–7

     

    Conventional In Gel Digestion of Proteins

    [Overnight general protocol  for all sample quantities] 

    Buffers and Solutions

    Gel fragment preparation

    • Excise protein bands. Cut each into 1 mm pieces. Place into a low-binding tube (LoBind, Eppendorf). Also cut out a gel piece from a protein-free region as control.
    • Wash gel pieces with >10 volumes of Millipore water [~200µl] for 30 seconds, to wash out acetic acid.

    Destaining

    • For Coomassie Blue staining, destain two times for 5 minutes or until colorless with 200µl 100 mM (NH4)HCO3 / 50% Methanol (discard supernatants). Dehydrate for 5 minutes with 200µl 25 mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. Remove the solutions and discard. The gel pieces shrink and become white.
    • For Non-Destructive Silver staining, destain twice for 2 minutes or until colorless with 200µl of freshly prepared 1:1 solution of 100mM Sodium Thiosulfate [Na2S2O3] and 30mM Potassium Ferricyanide [K3Fe(CN)6]. Stop the reaction and wash out silver ions twice for 2 minutes with 500µl of Millipore water. Dehydrate for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  The gel pieces shrink and become white.
    • For Sypro Ruby staining, no destaining necessary, Dehydrate for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.  Remove the solutions and discard. The gel pieces shrink and become white.

       

    • Dry gel particles for 2 minutes in a vacuum centrifuge.

    Reduction and Alkylation

    • Rehydrate gel pieces in 50µl of freshly prepared 25mM Dithiothreitol (in 25mM (NH4)HCO3). Reduce the proteins for 20 minutes at 56°C.
    • Cool the gel to room temperature, pipet off any residual liquid and add 50µl of freshly prepared 55mM Iodoacetamide (in 25mM (NH4)HCO3). Alkylate the proteins for 20 minutes at room temperature in the dark.
    • Pipet off the liquid and wash gel pieces with > 20 volumes of Millipore water [~400µl] for 30 seconds to remove any residual Iodoacetamide. Dehydrate gel pieces for 5 minutes with 200µl 25mM (NH4)HCO3 / 50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.
    • Dry gel particles for 2 minutes in a vacuum centrifuge.

    Trypsin Digestion

    • Rehydrate gel pieces for 5 minutes at room temperature in 20µl [10ng/µl] Trypsin (Promega Sequence Grade Modified) in 25mM (NH4)HCO3 / 3% Acetonitrile [ pH =~8.5].
    • Overlay the rehydrated gel particles with a minimum amount of 25mM (NH4)HCO3 to keep them immersed throughout the digestion. Incubate 16-24 hours at 37ºC.

    Peptide Recovery

    • Extract digested peptides with 50µl Millipore water / 1% TFA by vortexing 10 minutes at room temperature (max speed). Transfer solution to a new low-binding tube (LoBind, Eppendorf).
    • Perform an additional extraction with 80µl of 70% ACN / 25% H2O / 5%TFA.
    • Dry peptide solution completely in a vacuum centrifuge ( 1-2 hours).
    • Reconstitute peptides in 20µl of Millipore water / 0.1% TFA by incubating for 5 minutes at room temperature with intermittent vortexing.
    • Sample is ready for LC-based analysis.
    • Perform ZipTip-C18 column (Millipore) clean-up before spotting onto MALDI plate.

    References

    • Jimenez, C.R. Current Protocols in Protein Science. 1998; 16.4.1-16.4.5
    • Gharahdaghi, F. et al. Electrophoresis.1999; 20: 601-605.
    • Hirouki, K. et al. Rapid Commun. Mass Spectrom. 2001: 15; 1416-1421.