In-gel tryptic digests of 1-dimensional (1-D) or 2-dimensional (2-D) acrylamide gels are commonly used in proteomics to identify unknown proteins of interest. The unknown proteins are excised from the gel, digested with trypsin then analyzed via MALDI-MS or HPLC-MS with subsequent identification via bioinformatics (1).
The basic elements of the procedure include destaining, reduction/alkylation, in-gel digestion, extraction and analysis.
Trypsin is typically the enzyme of choice for the digestion step. Recommended digestion conditions range between several hours to overnight. Following digestion, the peptides are extracted and dried. An outline of example digestion/extraction protocol is follows:
- Overnight digestion with trypsin at 37°C.
- Extract the gel slice twice with 50% ACN/%5 TFA for 60 minutes.
- Pool the extracts and dry in a Speed Vac® at room temperature for 2 hours.
- Mass Spec analysis.
Using a combination of ProteaseMAX™ Surfactant and trypsin protein digestion and peptide recovery are completed in single step as short as 1 hour. During the digestion the surfactant enhances peptide extraction eliminating the need for post-digestion extraction. In gel digestion data quality is improved by the recovery of longer peptides that typically remain trapped in the gel under conventional conditions.
Overview of in-gel tryptic digestion using ProteaseMAX™ Surfactant:
- Digestion for 1 hour at 50°C or 2 hours at 37°C using the appropriate amount of trypsin and 0.01% ProteaseMAX™ Surfactant.
- Collect the condensate from the tube walls by brief centrifugation
- Add TFA to 0.05% to inactivate the trypsin.
- Peptides can directly analyzed with MALDI or concentrated using C18 ZipTip® tips.
- For LC-MS we recommend centrifuging the digest at 12,000-16.000 x g to remove particulate material and degraded ProteaseMAX™ Surfactant.
View a comparison of overnight trypsin digestion to the 1-hour digestion using ProteaseMAX™ Surfactant.
- Rosenfeld, J. et.al. (1992) Anal. Biochem, 203(1) 173-9.PubMed.
Latest posts by Gary Kobs (see all)
- Mutation Analysis Using HaloTag Fusion Proteins - March 11, 2019
- Optimizing Pressure Cycling Sample Preparation for Bottom-Up Proteomics - February 11, 2019
- Understanding Mechanisms of Pesticide Resistance to Thiamethoxam in the Cotton Aphid - January 11, 2019