Intact protein/peptide analysis
In-gel tryptic digestion of proteins
Intact protein/peptide analysis top
Analysis of an intact, non-digested protein or peptide sample by electrospray ionization mass spectrometry ( ESI-MS) can provide confirmation that a gene product of interest has been biosynthesized and isolated. This measurement can also provide information about the presence of degradation products, amino acid substitutions, bioconjugation products, and post-translational modifications such as phosphorylation or methylation. Sample preparation is crucial for obtaining a high-quality protein mass spectrum.
- Target analyte concentration: In the range 3 to 50 pmol/µL
- Typical solution composition: Water/organic solvent (1:1) with organic acid (0.1% to 1%)
- Water: Distilled, deionized water.
- Organic solvent: Typically acetonitrile. Other solvents such as methanol, ethanol, n-propanol, isopropanol, etc., will also suffice. The solvent should be HPLC grade. The exact ratio of water to organic solvent is not critical, although 1:1 is typical.
- Organic acid: Formic acid or acetic acid.
- Minimum sample volumes
- Syringe infusion electrospray: At least 100 microliters.
- Nanoelectrospray: At least 5 microliters.
- Interfering contaminants – Avoid or remove from sample
- Salts
- Metal cations: Li+, Na+, K+, Ca2+, Mg2+, etc.
- Inorganic anions such as phosphate, sulfate, and halides.
- Alkylammonium salts.
- Guanidinium salt
- Detergents/stabilizers: For example, SDS, PEG, PPG, Tween, CHAPS, Triton, and urea.
- Buffers: For example, HEPES, PBS, MES, MOPS, and Tris.
- Acceptable buffers – Use only if necessary (at concentrations ≤ 100 mM)
- Ammonium acetate
- Ammonium bicarbonate
- Ammonium formate
- Useful sample clean up tools
In-gel tryptic digestion of proteins top
Guidelines to minimize keratin contamination
- Perform as much of the work as possible within a laminar flow hood or a biological safety cabinet (BSC).
- A clean-room lab coat, nitrile or vinyl gloves and, if necessary, sleeve protectors should be worn.
- The inside of the BSC should be wiped down with water and ethanol prior to beginning work.
- All containers, tools, and apparatus should be wiped down with water and ethanol prior to placing them in the BSC.
- Previously opened containers should not be opened within the BSC.
- Do not use latex gloves or tubing.
Solutions
- 25 mM ammonium bicarbonate (aqueous)
- 25 mM ammonium bicarbonate in 1:1 acetonitrile/water
- 45% water/50% acetonitrile/5% formic acid
- 12.5 ng/µL (525 fmol/µL) trypsin in 25 mM ammonium bicarbonate (aqueous)
Procedure
- Excise the stained bands of interest from the gel using a scalpel or razor blade.
- Dice each gel slice of interest into small pieces (approximately 1 mm 2) using a scalpel or razor blade and add into a 0.65 mL siliconized tube.
- Add enough 25 mM ammonium bicarbonate in 1:1 acetonitrile/water to fully immerse the gel pieces and vortex for 10 minutes.
- Remove the supernatant using a gel-loading pipette tip and discard.
- Repeat steps 3 and 4 up to three times.
- Vacuum centrifuge the gel pieces to complete dryness (~20 to 30 minutes).
- Reduce and alkylate (optional - recommended for disulfide-rich proteins).
- Prepare fresh solutions.
- 10 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate with 10% acetonitrile
- 55 mM iodoacetamide in 25 mM ammonium bicarbonate (aqueous)
- Add enough 10 mM dithiothreitol solution to fully immerse the gel pieces and vortex and centrifuge briefly.
- Incubate at 56 °C for one hour.
- Cool to room temperature and remove and discard the supernatant.
- Add enough 55 mM iodoacetamide solution to fully immerse the gel pieces and vortex and centrifuge briefly.
- Incubate in the dark at room temperature for 45 minutes.
- Remove and discard the supernatant.
- Add enough aqueous 25 mM ammonium bicarbonate to fully immerse the gel pieces and vortex and centrifuge briefly.
- Remove and discard the supernatant.
- Add enough 25 mM ammonium bicarbonate in 1:1 acetonitrile/water to fully immerse the gel pieces and vortex and centrifuge briefly.
- Remove and discard the supernatant.
- Repeat steps h to k.
- Vacuum centrifuge the gel pieces to complete dryness (~20 to 30 minutes).
- Add one volume of the trypsin solution. This volume can be estimated from the volume of the excised gel band. For example, 2 mm × 5 mm × 1 mm = 10 mm 3 = 10 µL.
- Incubate on ice or at 4 °C for 30 minutes.
- Remove and discard any excess trypsin solution remaining after 30 minutes.
- If necessary, add a minimum amount of aqueous 25 mM ammonium bicarbonate to keep the gel pieces hydrated during digestion.
- Centrifuge briefly and incubate at 37 °C for 6 to 8 hours.
- Add a volume of water to the digest equal to two or three times the volume of the excised gel piece, vortex for 10 minutes, sonicate for 5 minutes, and centrifuge briefly.
- Extract the supernatant and transfer it into a fresh centrifuge tube (Tube I; do not discard).
- Add enough 45% water/50% acetonitrile/5% formic acid to the tube containing the gel pieces so that the pieces are fully immersed, vortex for 10 minutes, sonicate for 5 minutes, and centrifuge briefly.
- Extract the supernatant and add it into Tube I.
- Repeat steps 15 and 16 twice.
- Reduce the volume of Tube I to 10 µL using a vacuum centrifuge prior to submitting samples for analysis by LC-MS.
References
- Rosenfeld, J.; Capdevielle, J.; Guillemot, J. C.; Ferrara, P. “In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.” Anal. Biochem.1992, 203, 173-179.
- Hellman, U.; Wernstedt, C.; Gonez, J.; Heldin, C.-H. “Improvement of an ‘in-gel’ digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing.” Anal. Biochem.1995, 224, 451-455.
- Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J.V.; Mann, M. “In-gel digestion for mass spectrometric characterization of proteins and proteomes.” Nature Protocols.2006, 1, 2856-2860.
- Biringer, R. “Protocol for a keratin-free environment.” Thermo Electron Corp. (accessed September 12, 2007).
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