In-gel digestion followed by mass spectrometric analysis is a widely used technique for protein identification. This method involves separating proteins by electrophoresis on a polyacrylamide gel, followed by excision of the protein bands of interest. The proteins are then subjected to enzymatic digestion in the gel, typically with trypsin, which cleaves the protein into smaller peptides.
The resulting peptides are then extracted from the gel and analyzed by mass spectrometry. In this analysis, the peptides are ionized and separated by their mass-to-charge ratio (m/z) in the mass spectrometer. The resulting mass spectra can be used to identify the peptides and, subsequently, the proteins from which they originated.
Database searching software is commonly used to match the observed peptides to a protein database. This allows for the identification of the proteins present in the original sample. The accuracy of protein identification by this method is greatly improved by the use of high-resolution mass spectrometry and the availability of comprehensive protein databases.
In-gel digestion and mass spectrometric analysis is a powerful tool for the identification of proteins in complex mixtures. This technique has been used in a variety of fields, including proteomics, drug discovery, and biomarker identification.
1. SDS Acrylamide Gel
Use of a shaking platform allows more efficient gel washing for these steps.
a) Run 1-D or 2-D SDS acrylamide gel under conditions suitable for proteins of interest.
b) Wash gel in 200 mL water for 5 min. Repeat three times.
c) Visualize proteins by staining gel in SYPRO fluorescent dye or 0.1% colloidal Coomassie Blue for approx 1 h.
d) De-stain in water for 1–2 h.
2. In-Gel Digestion
More efficient removal of buffer and solvent from the gel pieces can be achieved with a P1000 pipet tip with a P10 tip stuck on the end.
a) Excise the stained gel band (identified by SYPRO or colloidal Coomassie Blue) by cutting out center (most concentrated part of band) to minimize the amount of acrylamide.
b) Incubate three times (in approx 200 μL 0.2M NH4HCO3/50% acetonitrile) for 30 min each at 30°C to remove the SDS.
c) Incubate the gel band in DTT (20 mM) in 200–300 μL of 0.2 M NH4HCO3/50% aqueous acetonitrile for 1 h at 30°C to reduce the proteins.
d) Wash three times in approx 200 μL 0.2 M NH4HCO3/50% acetonitrile.
e) Alkylate cysteine residues in fresh iodoacetamide (50 mM) in 100 μL, 0.2 M NH4HCO3/50% acetonitrile for 20 min at room temperature in the dark.
f) Wash three times in 500 μL 20 mM NH4HCO3/50% acetonitrile.
g) Cut the band into 1 × 2 mm pieces.
h) Centrifuge for 2 min at 10,000g (or top speed) in a microcentrifuge.
i) Cover pieces with acetonitrile (they must turn opaque white).
j) Dry the gel band completely by centrifugal lyophilization in centrifugal evaporator (approx. 30 min).
k) Rehydrate gel band with trypsin solution. Use approx 0.5–1.0 μg trypsin freshly made up in 60 μL 50 mM NH4HCO3, for 15–30 min at 4°C.
l) Add sufficient digestion buffer to make up to about 100 μL, i.e., enough to cover the gel pieces.
m) Incubate at 32°C, overnight—i.e., approx 16 h.
3. Peptide Extraction
a) The following day, centrifuge for 2 min at 10,000g (or top speed) in microcentrifuge.
b) Collect the digest buffer from above the gel pieces.
c) Add 100–200 μL 50% acetonitrile to the gel pieces, sonicate in a sonic bath for approx. 30 min (at 35–40°C), leave 1 h, centrifuge as before, and collect the supernatant.
d) Dry the acetonitrile extract to approx 20–50 μL in the centrifugal evaporator, then combine with the aqueous extracts.
e) This gives a total volume of approx 100–150 μL. Store at –20°C if not analyzed by MS immediately.
f) Desalt peptides for mass fingerprinting and/or sequencing by tandem ESMS or MALDITOF MS. For nanospray on an ion-trap mass spectrometer, use the protocol described in 4.
4. Peptide Identification by Nanospray MS Analysis of Smaller Proteins (<60 kDa)
a) For nanospray MS, fit the C8 reverse-phase column (0.8 mm × 2 mm) with PTFE tubing (3.5 cm) at the inlet end to allow syringe needle to fit snugly. Fit the outlet end with 5.5 cm of 50-μ fused silica tubing (see Note 6) to allow this to insert into nanospray needle.
b) Wash the C8 reverse-phase column (0.8 mm × 2 mm) with 200 μL formic acid (0.01% in 95% acetonitrile).
c) Equilibrate the column with approx 200 μL aqueous formic acid (0.01%).
d) The sample (dissolved in approx 20 μL aqueous formic acid, 0.01%) is loaded slowly onto the column using a 25-μL Hamilton syringe. If the solution is more dilute (i.e., if more sample is required to give good spectra), load up to 3 × 25 μL.
e) Wash column with 15 μL aqueous formic acid (0.01%).
f) Elute directly into the nanospray needle with aqueous formic acid (0.01%) containing 60% methanol to allow 2 μL to enter needle.
g) After use, store column in 95% aqueous acetonitrile.
5. Nanospray MS Analysis of Larger Proteins (>60 kDa)
a) Follow Subheading 4., steps 1–5
b) Elute directly into the nanospray needle with 2 μL aqueous formic acid (0.01%) containing 20% methanol, then in 10% incremental steps of methanol to allow 2 μL to enter needle at each step.
c) If protein is large (i.e., >200 kDa) start with aqueous formic acid (0.01%) containing 10% methanol, then elute in 5% incremental steps of methanol.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.