In-gel digest (Coomassie stained) with trypsin - Proteomics

Proteomic protocols for mass spectrometry
In-gel digest (Coomassie stained) with trypsin
Adapted from:
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing
of proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850-858.
Chemicals:
• NH4HCO3: M = 79 g/mol; 50mmol/l = 4mg/ml
• Dithiothreitol (DTT): M = 154 g/mol; 10mmol/l = 1.54mg/ml
• Iodacetamide (IAA): M = 185 g/mol; 55mmol/l = 10.2mg/ml
• Trypsin: c = 20 – 25ng/µl (Promega)
c = 5ng/µl (Roché)
Excision of protein bands from polyacrylamide gels
• Wash the gel with water (2 changes, 10 minutes each)
• Excise the spots of interest from the gel. Cut the excised piece into 1 mm-cubes. See
general remarks.
• Transfer to a 0.5 µl microfuge tube
Washing of gel pieces
• Wash the gel particles with water and 50 mM NH4HCO3/acetonitrile 1+1 (v/v) for 15 min
• Remove all remaining liquid and add enough acetonitrile to cover the gel particles. The
gel plugs shrink and stick together.
• Remove the acetonitrile.
• Rehydrate the gel pieces in 50 mM NH4HCO3.
• After 5 min add an equal volume of acetonitrile.
• Remove all liquid after 15 min of incubation.
• Add enough acetonitrile to cover the gel particles.
• After the gel pieces have shrunk remove the acetonitrile.
• Dry down gel particles in a vacuum centrifuge.
Reduction and alkylation
• Swell the gel particles in 10 mM dithiotreitol/50 mM NH4HCO3 (freshly prepared).
• Incubate for 45 min at 56°C.
• Chill tubes to room temperature.
• Remove excess liquid and replace it quickly by roughly the same volume as above of
freshly prepared 55 mM iodoacetamide (light sensitive!) in 50 mM NH4HCO3.
• Incubate for 30 min at room temperature in the dark.
• Remove iodoacetamide solution.
• Wash the gel particles with 50 mM NH4HCO3 and acetonitrile (1+1; v/v), one or two
changes each, 15 min per change.
• Add enough acetonitrile to cover the gel particles.
• After the gel pieces have shrunk remove the acetonitrile.
• Dry down the gel particles in a vacuum centrifuge.
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Proteomic protocols for mass spectrometry
In-gel digestion
• Add just enough freshly prepared enzyme solution (in 25 mM NH4HCO3 ) to cover the
gel.
• Incubate at 37°C for 30 minutes.
• Remove excess enzyme solution.
• Add enough 25 mM NH4HCO3 (approx. 2-3µl) to keep the gel wet overnight, but avoid
excess liquid.
• Incubate at 37°C overnight.
Extraction of peptides
• Apply extraction buffer enough to cover the gel plugs completely, but avoid too much of
excess volume (dilution of sample!). Support of the extraction by ultrasonication for a few
minutes may improve the extraction yields. Recommended time frame for extraction:
30min at room temperature.
• The optimum composition of the extraction buffer will depend on the nature of the protein
digested and on the aim of analysis respectively, and, to certain extent, on the MALDI
preparation protocol you are going to use. For a number of applications, application of
only one extraction buffer of a certain composition (ranging from 100/0 to 50/50 TF
enough A 0.1% / ACN) may be sufficient, f.i. for many routine protein identification
tasks. However, stepwise extraction using buffers of increasing ACN content may
improve the overall sequence coverage achieved in the following MALDI-TOF analysis.
Keep in mind also, that some prep protocols, f.i. Anchorchip Thin Layer (ACTL), require
the absence of any organic solvent in the peptide solution. Applying this preparation
protocol, you have the choice: either you extract using a buffer containing ACN, which
forces you to remove it from the extract prior to MALDI preparation, or you avoid any
ACN in the extraction buffer, which allows you to save the subsequent evaporation step,
but may lead to a certain under-representation of those peptides which are less soluble in
pure aqueous solvent.
Mass spectrometric peptide analysis
• If purification is necessary, perform ziptip or microcolumn purification. Alternatively,
Anchorchip Thin Layer (ACTL) is a MALDI prep protocol, which is able to treat
contaminated samples in a very efficient manner. Please refer to the Anchorchip manual
in this matter.
General remarks
Size of gel pieces: Cut as close to the protein band as possible to reduce the amount of
"background" gel. Avoid to use gel pieces much bigger than 1 mm x 1 mm.
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Proteomic protocols for mass spectrometry
Excise a gel piece of roughly the same size from a non-protein containing region of the gel for
use as a control.
Solvent amounts: Unless otherwise specified all the solvent volumes used in the washing
steps should roughly equal twice the gel volume.
Solvent Purity: All solvent should be as pure as possible. Make sure that the bottles remain
close when not in use.
Lab materials purity: Scalpel, pipette tips and tubes should be clean. Use original Eppendorf
tubes.
Contamination by ceratin: Ceratin contamination should be avoided. Avoid ceratin to fall on
the gel, in bottles or on pipette tips. It might help to use clean gloves (washed with water).
Prepare the trypsin solution: Sequencing grade, modified trypsin is recommended. The
solid powder is solved in 1 mM hydrochloric acid (for Roche Diagnostics, Mannheim trypsin)
or 50 mM acetic acid (for Promega trypsin) giving a concentration of 100 ng/µl. Store 10 µl
aliquots at -20°C.
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