This is because one of the most time-extensive steps in the journey to characterize proteins has been reduced from 2 days to 60 mins for a large protein such as immunoglobulin G (IgG) pictured in this post!
Need to Breakdown Proteins to Peptides
Over the years, I’ve listened to many protein chemists expound on trypsin digestion (link to typical 2-day protocol pdf) as one of the most time-intensive steps in the journey to characterize proteins. The need to break down the protein of interest into understandable peptides (YouTube video of trypsin digestion) is critical to the study of its structure and function. Well over 90% of all peptide mapping assays use trypsin digestion due to its well-defined specificity. Trypsin (Wikipedia link) hydrolyzes only the peptide bonds in which the carbonyl group is followed either by an arginine (Arg) or lysine (Lys).
You might find this 60-min on-demand webinar, titled, Sample Preparation Technologies for Improved Peptide Quantitation Workflow, (link to registration page; you will need to register before playing the webinar) on workflows that deliver high levels of data quality while balancing the needs of sample throughput and regulatory compliance helpful.
What Does a Faster Protein Digestion do for Me Besides Saving Time?
Peptide mapping is now amenable to high-throughput workflow. This new technology comes as a complete kit with buffers, reagents and a collection plate, and a choice of sample clean-up, from an optional filter or solid-phase extraction plate (for clean-up/pre-concentration). This workflow allows for simple, fast, generic, and robust analytical methods within a high-throughput biopharmaceutical environment.
Application Note 21198, Improvement in Speed and Reproducibility of Protein Digestion and Peptide Quantitation, Utilizing No… (link to downloadable pdf), demonstrates the high-throughput, reproducible workflow that can be applied to non-targeted, semi-targeted, or targeted quantitative environment for peptide quantificaiton and analysis.
What About the Reproducibility of this Technique?
Trypsin activity varies significantly depending on the buffer used and additives such as guanidine HCl and urea have a concentration-dependent adverse effect on the digest efficiency. Furthermore, buffer species such as phosphate buffer saline (PBS) and ammonium bicarbonate (ABC) are also found to adversely affect digestion. These factors often result in varied digestion efficiency and non-reproducible results.
Application Note 21179, Selecting Buffers to Remove Uncertainty in Tryptic Digestion (link to downloadable pdf) demonstrates how protein digestion kits remove uncertainty associated with conventional solution-based tryptic digestion protocols, resulting in higher reproducibility and sample characterization in peptide quantification.
Applications and Resources
Check out the following article and applications for additional information on trypsin digestion and peptide quantification and mapping:
- R.H. Erickson and Y.S. Kim (1990): Digestion and Absorption of Dietary Protein. Annual Review of Medicine41, pp. 133-139 .
- Column Temperature Control in Peptide Mapping (application in AppsLab)
- High Resolution Peptide Mapping for Biopharmaceutical Analysis (application in AppsLab)
- Improved analysis of four peptide maps on a Thermo Scientific Acclaim 120 C18 HPLC column (application in AppsLab)
- Reliable Results in Peptide Mapping Using the Vanquish Flex UHPLC System (application in AppsLab)
- Increased resolution of tryptic digests of proteins using a Thermo Scientific Acclaim HPLC column (application in AppsLab)
Chromatography and Software Analytical Solutions
The following resources could prove useful if you working in the field of proteomics research:
- Our Protein Identification Software: Our protein identification and characterization software (Thermo Scientific PepFindersoftware) provides accurate identification, in-depth characterization, and relative quantitation of biotherapeutic and other proteins from mass spectrometric data. In addition, it provides automated workflows for glycopeptide identification, disulfide bond mapping, and quantification of PTMs.
- Our bio-inert UHPLC systems have been built for biopharmaceutical development and provide faster separations and reproducible results.
I hope you’re as excited about this protein digestion breakthrough as I am. If the protein digestion kits are of interest to your laboratory we would like to hearaboutyour thoughts and experiences.