The
mass spectrometric identification and quantification of proteins
requires prior splitting of the proteins into peptides. This splitting
is called protein digestion. It is carried out with the aid of enzymes
such as the protease trypsin or endopeptidase Lys-C.
The analysis
of proteins by mass spectrometric methods can be carried out either with
proteins in solution or with proteins separated by SDS gel
electrophoresis. The sample preparation consists of several steps. After
denaturation and washing of the sample, the first step is the reduction
and subsequent alkylation of the sulfhydryl groups of the proteins. The
reagents TCEP and CAA are used for this purpose. This is followed by
the actual enzymatic digestion and subsequent extraction of the newly
formed peptides, which can then be analysed by mass spectrometry.
For
the generation of robust proteomic data, a reproducible protein
splitting process is necessary. Here, the use of automated sample
preparation robots is extremely useful.
The basis for this
automation is the CHRONECT Robotic Autosampler and the CHRONOS software
developed by Axel Semrau. It allows a time-optimized utilization of the
robot system and thus enables a high sample throughput.
Sequence of the automation
Protein digestion in solution (In-Solution Digest)
- Place protein sample in buffer
- Add TCEP to perform reduction / 30 min
- Alkylation with CAA / 20 min
- Add LysC and first digestion / 3 h
- Dilute sample with buffer
- Add trypsin and second digestion / 10 h
- Stop digestion by TFA
To
perform the digestion of proteins directly in the electrophoresis gel,
the CHRONECT Workstation Proteomics includes a special tray with
connection to a vacuum pump.
Protein digestion in electrophoresis gel (In-Gel Digest)
- Place gel section in tray with vacuum station
- Wash gel intensively
- Add TCEP to perform reduction / 30 min
- Alkylation with CAA / 20 min
- Add trypsin and digest / 10 h
- Stop digestion by TFA