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十九烷酰胺胶中肽提取和蛋清消化摄取技能,肽

文章作者:生命科学 上传时间:2019-08-27

大旨提醒:Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, t

主干提醒:Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, t

中央提醒:ABSTRACTAnalysis of cellular proteins by Mass Spectrometry requires the isolation of protein complexes, and e

Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimensional gel electrophoresis has provided a tool for differential protein display, which allows for the quantitative analysis of many of proteins simultaneously. Complex biological questions can now be approached by analyzing differences in the 2D gel patterns between control and experimental states.

Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimensional gel electrophoresis has provided a tool for differential protein display, which allows for the quantitative analysis of many of proteins simultaneously. Complex biological questions can now be approached by analyzing differences in the 2D gel patterns between control and experimental states.

ABSTRACT

While high sensitivity staining methods could visualize proteins at increasingly low levels, originally protein identification was limited by the sensitivity of Edman sequencing. However, in the last few years mass spectrometry has become established as a viable and more sensitive alternative, with the development of ESI-MS and MALDI-MS. Peptide mass fingerprinting uses the peptide masses obtained by digestion to search the protein and DNA databases, to find proteins that show a similar theoretical digest pattern .

While high sensitivity staining methods could visualize proteins at increasingly low levels, originally protein identification was limited by the sensitivity of Edman sequencing. However, in the last few years mass spectrometry has become established as a viable and more sensitive alternative, with the development of ESI-MS and MALDI-MS. Peptide mass fingerprinting uses the peptide masses obtained by digestion to search the protein and DNA databases, to find proteins that show a similar theoretical digest pattern .

Analysis of cellular proteins by Mass Spectrometry requires the isolation of protein complexes, and enzymatic digestion into peptides. After proteins are separated into bands through electrophoresis on an acrylamide gel, this protocol provides a method for reduction, alkylation and in-gel digestion into peptides, as well as extraction of the resultant peptides.

In the protocol below, we describe the in-gel digestion procedure, as it is routinely performed in our laboratory, for peptide mass mapping of picomole to subpicomole quantities of protein derived from coomassie- or silver stained polyacrylamide gels. First, SDS is removed from the gel prior to digestion, in several ammoniumcarbonate/acetonitrile washing steps. The excised gel pieces are subsequently dried and rehydrated with enzyme in buffer. Alternatively, the washed gel pieces may be reduced and S-alkylated prior to rehydration with enzyme. After digestion, the peptides are extracted from the gel and mass analyzed.

In the protocol below, we describe the in-gel digestion procedure, as it is routinely performed in our laboratory, for peptide mass mapping of picomole to subpicomole quantities of protein derived from coomassie- or silver stained polyacrylamide gels. First, SDS is removed from the gel prior to digestion, in several ammoniumcarbonate/acetonitrile washing steps. The excised gel pieces are subsequently dried and rehydrated with enzyme in buffer. Alternatively, the washed gel pieces may be reduced and S-alkylated prior to rehydration with enzyme. After digestion, the peptides are extracted from the gel and mass analyzed.

MATERIALS

In-gel digestion procedure

In-gel digestion procedure

Buffers, Solutions, and Reagents

Materials

25 mM NH4HCO3 in 50% acetonitrile

25 mM NH4HCO3, pH 8

5% TFA/ 50% acetonitrile

0.1 mg/ml trypsin in 25 mM NH4HCO3, pH 8

10 mM dithiotreitol in 25 mM NH4HCO3

55 mM iodoacetamide in 25 mM NH4HCO3

Generally, trypsin is the protease of choice for peptide mass fingerprinting, because of its reliability and its substrate specificity, yielding peptides with C-terminal basic residues , which facilitates ionization and subsequent mass spectrometric sequencing.

Dissolve trypsin just before use in ice-cold buffer .

  1. Excise protein bands/spots of interest from the gel and cut each gel piece into small particles using a scalpel and place into a 0.65 ml siliconized tube . Also cut out a gel piece from a protein-free region of the gel, for a parallel control digestion to identify trypsin autoproteolysis products.

A small gel particle size facilitates the removal of SDS during the washes, and improves enzyme access to the gel.

For a silver-staining protocol compatible with mass spectrometry, see Shevchenco et al. .

  1. Add ~100 ml 25 mM NH4HCO3 in 50% acetonitrile and vortex for 10 min. Use gel-loading pipet tips to remove the solution and discard. Repeat this wash/dehydration step up to ~2-3 times.

At this point, the gel slices shrink and become white. This visual criterium should be used to determine whether or not additional washes should be performed.

  1. Dry the gel particles for ~15 min in a vacuum centrifuge.

  2. Optional reduction and alkylation. Add 10 mM dithiotreitol in 25 mM NH4HCO3, enough to cover the gel pieces and reduce for 1 hr at 56 oC. Cool to room temperature and replace the DTT solution by roughly the same volume 55 mM iodoacetamide in 25 mM NH4HCO3. Incubate for 45 min at room temperature in the dark with occasional vortexing. Wash the gel pieces with ~100 ml 25 mM NH4HCO3 for 10 min while vortexing, dehydrate with ~100 ml 25 mM NH4HCO3 in 50% acetonitrile and rehydrate again with ~100 ml 25 mM NH4HCO3 and dehydrate again. Remove the liquid phase and dry the gel pieces in a vacuum centrifuge.

  3. Rehydrate the gel particles in 25 mM NH4HCO3, pH 8, containing 0.05 - 0.1 mg/ml trypsin by vortexing for 5 min. Do not add more solution than the amount that can be absorbed by the gel particles, otherwise a lot of trypsin autolysis will occur.

The enzyme-to-substrate ratio employed for in-gel digestions is greater than for in-solution digestions due to the hindered enzyme access to the protein substrate in the gel. Moreover, the relative low salt concentration of 25 mM is used to reduce the possibility of subsequent salt interference with ionization in the mass spectrometer. This concentration may be increased if poros microtips or ziptips are subsequently used for desalting.

  1. If neccessary, overlay the rehydrated gel particles with a minimum amount of 25 mM NH4HCO3, pH 8, to keep them immersed throughout digestion.

  2. Incubate 12-16 hours at 37 oC.

  3. To recover the peptides from the gel particles, perform ~3 extractions. For the first extraction, add 2 volumes of water and vortex for 10 min. For subsequent extractions, add 5% formic acid/50% acetonitrile. Use gel-loading tips to remove the peptide solution after each extraction and collect in a siliconized tube.

The siliconized microfuge tubes and the high formic acid concentration are used to minimize adsorptive sample loss. TFA may be used as an alternative for formic acid if MALDI-MS is used.

  1. Concentrate the recovered peptides by reducing the final volume of the extracts to ~10 ml in a vacuum centrifuge and add 5 ml 5% Formic acid/ 50% acetonitrile.

  2. Store the recovered peptides at -20 °C in slick tubes.

Materials

25 mM NH4HCO3 in 50% acetonitrile

25 mM NH4HCO3, pH 8

5% TFA/ 50% acetonitrile

0.1 mg/ml trypsin in 25 mM NH4HCO3, pH 8

10 mM dithiotreitol in 25 mM NH4HCO3

55 mM iodoacetamide in 25 mM NH4HCO3

Generally, trypsin is the protease of choice for peptide mass fingerprinting, because of its reliability and its substrate specificity, yielding peptides with C-terminal basic residues , which facilitates ionization and subsequent mass spectrometric sequencing.

Dissolve trypsin just before use in ice-cold buffer .

  1. Excise protein bands/spots of interest from the gel and cut each gel piece into small particles using a scalpel and place into a 0.65 ml siliconized tube . Also cut out a gel piece from a protein-free region of the gel, for a parallel control digestion to identify trypsin autoproteolysis products.

A small gel particle size facilitates the removal of SDS during the washes, and improves enzyme access to the gel.

For a silver-staining protocol compatible with mass spectrometry, see Shevchenco et al. .

  1. Add ~100 ml 25 mM NH4HCO3 in 50% acetonitrile and vortex for 10 min. Use gel-loading pipet tips to remove the solution and discard. Repeat this wash/dehydration step up to ~2-3 times.

At this point, the gel slices shrink and become white. This visual criterium should be used to determine whether or not additional washes should be performed.

  1. Dry the gel particles for 皇家赌场号hj85,~15 min in a vacuum centrifuge.

  2. Optional reduction and alkylation. Add 10 mM dithiotreitol in 25 mM NH4HCO3, enough to cover the gel pieces and reduce for 1 hr at 56 oC. Cool to room temperature and replace the DTT solution by roughly the same volume 55 mM iodoacetamide in 25 mM NH4HCO3. Incubate for 45 min at room temperature in the dark with occasional vortexing. Wash the gel pieces with ~100 ml 25 mM NH4HCO3 for 10 min while vortexing, dehydrate with ~100 ml 25 mM NH4HCO3 in 50% acetonitrile and rehydrate again with ~100 ml 25 mM NH4HCO3 and dehydrate again. Remove the liquid phase and dry the gel pieces in a vacuum centrifuge.

  3. Rehydrate the gel particles in 25 mM NH4HCO3, pH 8, containing 0.05 - 0.1 mg/ml trypsin by vortexing for 5 min. Do not add more solution than the amount that can be absorbed by the gel particles, otherwise a lot of trypsin autolysis will occur.

The enzyme-to-substrate ratio employed for in-gel digestions is greater than for in-solution digestions due to the hindered enzyme access to the protein substrate in the gel. Moreover, the relative low salt concentration of 25 mM is used to reduce the possibility of subsequent salt interference with ionization in the mass spectrometer. This concentration may be increased if poros microtips or ziptips are subsequently used for desalting.

  1. If neccessary, overlay the rehydrated gel particles with a minimum amount of 25 mM NH4HCO3, pH 8, to keep them immersed throughout digestion.

  2. Incubate 12-16 hours at 37 oC.

  3. To recover the peptides from the gel particles, perform ~3 extractions. For the first extraction, add 2 volumes of water and vortex for 10 min. For subsequent extractions, add 5% formic acid/50% acetonitrile. Use gel-loading tips to remove the peptide solution after each extraction and collect in a siliconized tube.

The siliconized microfuge tubes and the high formic acid concentration are used to minimize adsorptive sample loss. TFA may be used as an alternative for formic acid if MALDI-MS is used.

  1. Concentrate the recovered peptides by reducing the final volume of the extracts to ~10 ml in a vacuum centrifuge and add 5 ml 5% Formic acid/ 50% acetonitrile.

  2. Store the recovered peptides at -20 °C in slick tubes.

  • Acetonitrile
  • Ammonium bicarbonate , 100 mM
  • CaCl2, 1 M
  • Digestion buffer
    • 50 mM NH4HCO3, 5 mM CaCl2
  • Formic acid 90%, 5%
  • Milli-Q water
  • Iodoacetamide , 55 mM
  • K3Fe6
  • Na2S2O3·5H2O
  • Proteins in gel slices as obtained in Protocol 1, contained in a microfuge tube
  • Tris -phosphine hydrochloride
  • Trypsin, sequencing grade, modified

Special Equipment

  • Speed vacuum
  • Incubator, preset to 37°C

METHOD

Removal of Silver StainBefore the proteins are digested, the Coomassie or silver stain must be removed from the gel.

  1. If proteins were visualized by silver stain, first remove silver stain . However, if proteins were visualized by Coomassie, begin at step 4. If the silver-stained gel slice was stored in acetic acid, remove the storage solution and rinse the slice with water.
  2. For each gel slice, prepare a 30-mM solution of K3Fe6 and a 100-mM solution of Na2S2O3·5H2O. Mix 200 µl of each solution together and transfer the mixture to the microfuge tube containing the gel piece. Incubate for 5 min with gentle vortexing.
  3. Remove the liquid and wash each gel slice 3 times with 500 µl of water. Incubate the slices in 100 µl of 100 mM NH4HCO3 for 30 min. Discard the liquid and proceed with step 4.

Removal of Coomassie Staining and Treatment of Gel Slices

  1. At this point, the gel slices are either stained with Coomassie or have just had the silver stain removed. In either case, wash the gel slices with water for 15 min. All steps should be performed with light vortexing. The volume of solvents added in the wash and elution steps should be twice that of the cut gel pieces .
  2. Remove the liquid remaining from step 4 and add 100 µl of water, and then 100 µl of acetonitrile to the gel pieces. Incubate for 15 min at room temperature.
  3. Remove the liquid and add a sufficient volume of acetonitrile to cover the gel pieces . The pieces will dehydrate, shrinking and turning white and sticky. After this, remove the acetonitrile.
  4. Rehydrate the slices by adding 100 µl of 100 mM NH4HCO3. Wait 5 min, and then add 100 µl of acetonitrile. Incubate for 15 min at room temperature. If Coomassie staining was used to visualize the proteins and a strong blue color persists, incubate for a further 15 min with continuous gentle vortexing. Remove the liquid and dry down the gel pieces in a speed vacuum for 15 min.

Reduction, Alkylation, and In-gel Digestion

  1. Rehydrate the gel pieces using a sufficient volume of 10 mM TCEP in 100 mM NH4HCO3 to cover them . Incubate for 20-30 min at room temperature.
  2. Remove the liquid and quickly add the same volume of 55 mM IAA in 100 mM NH4HCO3. Incubate for 30 min at room temperature in the dark. It is important that the IAA is freshly prepared.
  3. Remove the liquid and dehydrate the gel by adding 100 µl of acetonitrile. After the gel pieces have shrunk and turned white and sticky, remove the acetonitrile. Add 100 µl of 100 mM NH4HCO3 and incubate for 5 min at room temperature to allow rehydration.
  4. Add 100 µl of acetonitrile to create a 1:1 mix of 100 mM NH4HCO3 and acetonitrile, and incubate for 15 min.
  5. Remove the liquid and dry the gel slices completely in a speed vacuum, for at least 30 min. Rehydrate in 100 µl of digestion buffer containing 12.5 mg/ml trypsin. Incubte at 37°C overnight.

Extraction of Peptides

  1. Centrifuge the trypsin digests briefly in a microcentrifuge and transfer the peptides to a 0.5-ml microfuge tube and reserve. In the meantime, add 100 µl of 25 mM NH4HCO3 to the gel pieces and incubate for 15 min. Then add 100 µl of acetonitrile and incubate for a further 15 min.
  2. Recover the liquid and transfer it to the 0.5-ml tube containing the peptides. Begin drying the peptides in a speed vacuum, while continuing with steps 14 and 15. Add 100 µl of 5% formic acid to the gel slices and incubate for 15 min. Next, add 100 µl of acetonitrile to the slices and incubate for 15 min.
  3. Recover the supernatant from the slices and transfer it to the 0.5-ml tube containing the peptides. Repeat step 14. Continue drying the sample in the 0.5-ml microfuge tube in the speed vacuum for ~2 hr, until there is only 10 µl remaining. At this point, the peptides are ready for analysis. Analysis can be done by ESI MS, ESI MS/MS , or MALDI/TOF.

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