Enhanced on-plate digestion of proteins using a MALDI-digestion chamber

- Utilizing a computer controlled humidity chamber for on-plate protein digestion.
- Reduction and Alkylation directly on the MALDI-target prior to digestion.
- Improvement of sequence coverage for on-plate digestion.
- Optimization of on-plate digestion duration and temperature.
- Improvement of on-plate digestion for small protein amounts.

A commercially available digestion chamber (SunDigest, SunChrom) is used for on-target digestion of proteins for subsequent direct MALDI-MS analysis. Temperature and humidity in the chamber can be controlled and kept constant during the reactions. For the first time, not only the tryptic digestion but also reduction and alkylation were performed in only one μL of sample directly on the MALDI sample plate. Each reaction step was optimized with regard to reactant concentration, temperature and time. Under optimized conditions a significant increase in sequence coverage is obtained. Proteins such as BSA and Apo-transferrin give best results when all reaction steps are performed at 50 °C, which results in a considerably reduced total time of 65 min compared to in-solution digests. Moreover, the on-target protocol is not only faster, but also superior with respect to the required protein amount; at the tested lower limit for PMF-analysis of 100 fmol the on-target procedure exhibits strongly superior sequence coverage and identification scores. Using lower protein amounts (10-50 fmol) an identification with MS/MS-analysis is possible. This newly developed protocol combines the benefits of the programmable SunDigest humidity chamber with a time-saving on-plate reduction, alkylation and digestion workflow for protein identification.

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