It’s time to analyze your protein and you are trying to decide where to begin. You are asking questions like: Which protease do I choose? How much enzyme should I use in my digest? How long should I perform my digest?
Unfortunately, there is no one-size fits all answer to this type of question other than… “well it depends.” All protease digests will be a balance between denaturing the protein sample to allow access to cleavage sites, optimizing conditions for the protease to function, and compatibility with your workflow and downstream applications. We provide general guidelines that work for most samples, but frequently you will need to optimize the conditions need for your specific sample and application.
Here, I use the example of a trypsin digest for downstream mass spectrometry to highlight key questions to ask and factors that can be optimized for any digest. Continue reading “What’s In YOUR Protein? Optimizing Protease Digestions to Get the Inside Scoop”
Trypsin/Lys-C Mix, Mass Spec Grade, is a mixture of Trypsin Gold, Mass Spectrometry Grade, and rLys-C, Mass Spec Grade. The Trypsin/Lys-C Mix is designed to improve digestion of proteins or protein mixtures in solution.It is a little known fact that trypsin cleaves at lysine residues with lesser efficiency than at arginine residues. Inefficient proteolysis at lysine residues is the major cause of missed (undigested) cleavages in trypsin digests.
Supplementing trypsin with Lys-C enables cleavage at lysines with excepetional efficiency and specificity. Following the conventional trypsin digestion protocol (i.e., overnight incubation at nondenaturing conditions, reduction,alkylation, 25:1 protein:protease ratio [w/w], mix and incubate overnight at 37°C.) Replacing trypsin with Trypsin/Lys-C Mix in this conventional protocol leads to multiple benefits for protein analysis including more accurate mass spectrometry-based protein quantitation and improved protein mass spectrometry analytical reproducibility.
In many proteome studies, sample analysis is performed directly from the liquid phase. This method avoids many of the time consuming steps associated with in-gel digestion. As with in-gel digestions, trypsin is the enzyme of choice. Trypsin cleaves at C-terminal of lysine (K) and arginine (R) amino acids resulting in a N-terminal amine group that can accept a proton and a basic side chain residue of the K/R that will also take up a proton. These charged particles can be easily ionized when analyzed by mass spectrometry.
Immobilized Trypsin provides a fast and convenient method for digesting a range of concentrations of purified protein, or complex protein mixtures. Continue reading “Enhanced In-Solution Tryptic Digestions: Immobilized Trypsin”
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: Continue reading “Streamlined in-gel digestion protocol using ProteaseMAX™ Surfactant, Trypsin Enhancer”
Protein biomarker discovery is an area of considerable current interest in biology and medicine.
The implementation of proteomics technology in the field of protein biomarker discovery has expanded in the past few years to enable the identification of biomarkers from a variety of biological samples including cell lysates, tissue samples, serum, plasma and urine (for a review refer to Gao, J. et al. (2005) Methods. 35, 291-302).
One method utilized for the discovery of protein markers includes the use of trypsin digestion of target proteins followed by the analysis of the resulting peptide fragments by mass spectrometry. Continue reading “The Search for Biomarkers: Beyond the Norm”