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Ahoy! Do I Need Satellite Navigation for my Peptide Map?

Team TFS
Team TFS

pepMy colleague Karen Thorsen’s recent blog post about super-fast protein digestion (link to blog post) got me thinking about the peptide mapping process, how hard it is, and how it could be made easier. And, perhaps more to the point, why is peptide mapping so important?

Turning to my trusty friend Wikipedia, a peptide (link to Wikipedia article) is “a biologically occurring short chain of amino acid monomers linked by peptide (amide) bonds.” So they’re pretty basic building blocks really, but the role that they play is incredibly significant to the production of those magical biotherapeutic proteins that are proving so good at helping patients. Time to learn a bit more…


Unfold the Map, Switch On the Sat-Nav, and Map Those Peptides!

The phrase “peptide mapping” really refers to identifying all of the peptides that go to make up a single protein. There are several stages during the life cycle of a biotherapeutic drug that require peptides to be mapped, and this mapping is done for different reasons. During the discovery and development phases of a biotherapeutic, it is necessary to understand the entire structure of the molecule, i.e. to characterize it. One way to do this is to study the molecule in an intact form, sometimes known as top-down characterization. However, this is seldom comprehensive, and so more commonly, the proteins are characterized by studying the component peptides, known (you guessed it!) as bottom-up characterization. It’s still quite a tricky procedure, and for full structural characterization, a powerful mass spectrometric technique, such as an Orbitrap™-based instrument, is usually used. There’s a good technical description of peptide mapping (link to PDF) produced by U.S. Pharmacopeia.

The other common peptide mapping application is used during clinical trials and batch testing, where the peptides are mapped to prove that this batch of protein is exactly the same (structurally) as the last one. In fact, peptide mapping is now considered a routine part of biotherapeutic characterization.

Is Peptide Mapping Regulated?

Clearly, the regulating authorities are going to have something to say about the peptide structure of a protein if it could affect the way the drug works. For example, the European Medicines agency (link to EMA PDF), when referring to Structural Characterisation and Confirmation, has the following to say: “Peptide mapping (under reducing and non-reducing conditions), mass spectrometry, or other appropriate techniques may be useful for this evaluation.”

Similarly, the U.S. FDA (link to PDF about test procedures for biological products) does talk of peptide mapping being important, stating that “…the resulting peptide fragments are analysed by high performance liquid chromatography (HPLC) or other appropriate analytical procedures. The peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing, or mass spectrometry.”

Suffice to say that peptide mapping is a must-do if you are currently developing a new biopharmaceutical.

What About the Instruments?

Working as I do for a vendor of scientific instruments, I found it beneficial to take a look at what analytical capabilities are useful to make peptide mapping a little easier. Choice of liquid chromatograph (or more specifically, UHPLC) is really significant, with reproducibility being just as important as high resolution. Our latest chromatography system (link to HPLC 2015 video ), the Thermo Scientific™ Vanquish™ Flex UHPLC, is particularly well-suited to this role, with some pretty impressive data (link to application note PDF). The image below shows five overlaid chromatograms for consecutive injections. The consistency is excellent, and gives the peptide mappers the all-important confidence. For reference, this new peptide mapping column (link to product page) is a Thermo Scientific™ Acclaim™ RSLC column. The background information relating to this figure is available in the application note.



There’s obviously a part to play for techniques that can determine further detail, mass spectrometry being the most obvious. In fact, it is arguable that to obtain the best fine-structure information, mass spectrometry is not just useful but essential. Industry contacts tell me that during the discovery and development stages, MS is used routinely, but once a drug is being manufactured, routine batch testing may only require UHPLC – often considered an easier-to-use technique than mass spectrometry.

…And Does Software Play A Role?

One aspect of peptide mapping is that it produces a large amount of data. The chromatographic spectra are complicated, and ideally each peptide should be individually identified. Some companies have produced dedicated software that allows you to model the peptide sequence, which is a good way of confirming that what you are seeing is what the model would predict. Our own peptide mapping software (link to product page) has proved immensely popular, and we consider it an integral part of the peptide mapping workflow. Here’s a great video to give you a taste of what it is all about.


Additional Resources

Visit the resources below to learn more about peptide mapping.




Do you have any insight into peptide mapping and the associated challenges? Or, do you have an interesting story about the regulated environment that these drugs are produced in? If so, please let us know.