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

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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Version 1.0, 6.12.2000