Critical Considerations and Actual Trends
Some major drawbacks of the common protocols for the in-gel digestion are the extended time need and the multiple processing steps making the method error-prone in respect to contaminations (especially keratin). These disadvantages were largely removed by the development of optimised protocols and specialised reaction tubes.
More severe than the difficulties with handling are losses of material while processing the samples. The mass spectrometric protein analysis is often performed at the limit of detection, so even small losses can decide about success or failure of the whole analysis. These losses are due to washout during different processing steps, adsorption to the surface of reaction tubes and pipette tips, incomplete extraction of peptides from the gel and/or bad ionisation of single peptides in the mass spectrometer. In dependence of the different physicochemical properties of the peptides the losses can vary between 15 and 50%. Due to this heterogeneity of the peptides up to now a universally valid solution for this major drawback of the method has not been found.
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