The in-gel digestion step is a part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis.
Although the sensitivity of the method is significantly lower, the use of Coomassie is more common for samples destined for mass spectrometry since the silver staining impairs the analysis.
[8] At the same time, the ionic part of the solution diminishes the electrostatic bonds between the dye and the positively charged amino acids of the protein.
In course of the subsequent irreversible alkylation of the SH groups with iodoacetamide the cysteines are transformed to the stable S-carboxyamidomethylcysteine (CAM; adduct: -CH2-CONH2).
Reduction and alkylation of cysteine residues improves peptide yield and sequence coverage and the identification of proteins with a high number of disulfide bonds.
[14][15] Due to the rareness of the amino acid cysteine for most of the proteins the step of r&a does not effect any improvement of the mass spectrometric analysis.
[23][24] Nowadays most suppliers offer modified trypsin where selective methylation of the lysines limits the autolytic activity to the arginine cutting sites.
For the use of trypsin as protease and a temperature of 37 °C the time of incubation found in most protocols is 12-15 h. However, experiments about the duration of the digestion process showed that after 3 h there is enough material for successful mass spectrometric analysis.
[10] To meet the requirements of peptides with different physical and chemical properties an iterative extraction with basic or acidic solutions is performed.
[10] Many protocols contain an additional fraction of acetonitrile to the extraction solution which, in concentrations above 30% (v/v), is effective in reducing the adsorption of peptides to the surface of reaction tubes and pipette tips.
Some major drawbacks of the common protocols for the in-gel digestion are the extended time needed and the multiple processing steps, making the method error-prone with respect to contaminations (especially keratin).
Due to the highly time-consuming and work-intensive standard procedure, the method of in-gel digestion was limited to a relatively small number of protein spots to be processed at a time.
The advantages of the automation other than the larger number of spots to be processed at a time are the reduced manual work and the improved standardisation.
Due to the many handling steps of the method, the results of the manual process could vary depending on the dexterity of the user and the risk of contamination is high.
This lengthy procedure prevents the researcher from spontaneous identifications of a few interesting spots from a single gel as well as the need to operate the systems at full capacity.
Most of the kit systems are mere collections of the chemicals and enzymes needed for the in-gel digestion whereas the underlying protocol remains unchanged from the manual standard procedure described above.
The advantage of these products for the inexperienced customer lies in the guaranteed functioning of the diverse solutions in combination with a ready-made protocol for the process.
A few companies have tried to improve the handling process of in-gel digestion to allow even with manual sample preparation an easier and more standardised workflow.
The Montage In-Gel Digest Kit from Millipore is based on the standard protocol, but enables processing of a large number of parallel samples by transferring the handling of the gel pieces to a modified 96 well microplate.