I finished my PhD and began my career as an R&D scientist at the start of the millennium, when proteomics was in its infancy. In those days, peptide mapping was all the rage as researchers looked to break proteins down into their component parts for subsequent identification by mass spectrometry. I’ve subsequently changed roles during the past 15 years, not losing touch with proteins, but with peptide mapping to a large degree. Recently, peptide mapping has come back on my radar and I’ve seen some major advances in the technology to improve the process during that 15-year gap, which I will outline in this post.
Today, peptide mapping is used extensively in a number of fields, such as in proteomics to identify unknown proteins and potential biomarkers, as well as in biopharmaceuticals to confirm the sequence of the manufactured protein to ensure no changes have occurred compared to the original sequence.
The Peptide Mapping Workflow
Peptide mapping was first discovered and described in 1993, and the overall workflow has not changed much since this time. The first step is to break down the protein(s) into peptides. This is usually achieved by incubating the protein with a protease such as trypsin. These proteases cleave the protein into peptides at specific sequences/sites. The resulting peptides are then subjected to HPLC to separate them. The workflow can then take two different paths:
- If you are simply looking to confirm the sequence of a known protein, are confident in the retention time precision of your HPLC instrument, and have a control sample to compare the pattern to, then you can simply detect the peptides using UV and match this to the control to confirm the sequence.
- On the other hand, if you have unknown proteins you wish to identify or want to investigate post-translational modifications, then you would need to detect these on a mass spectrometer to get the accurate mass of the peptides. These identified peptides would then be matched against a database of known peptide masses, using software to indentify the peptides and subsequent protein.
Peptide Mapping in the Noughties
When I first encountered peptide mapping the process was relatively laborious, not always very accurate and predominantly used to identify unknowns. Proteins were typically contained within polyacrylamide gels and first had to be extracted or released from the gels before digestion into peptides. Trypsin was usually the protease of choice, but it was a long process (most people would leave the reaction overnight and for at least four hours at a minimum); it was not always 100% efficient, and reproducibility was a big problem. The HPLC separation was adequate, but retention time precision was often not good enough for confirmation with UV detection alone. The separation was however good enough if mass spectrometry was used for detection, if a little slow in the days before UHPLC. Mass spectrometry identification was typically performed on MALDI-TOF instruments and early versions of protein identification software and associated databases.
If I were to summarise my early experiences with peptide mapping, it was possible to obtain reasonably good results using these methods, but it was a slow process, the results were not always that reproducible and the instrumentation and software were relatively complex to use. Fast forward to 2016…
Peptide Mapping Today
Today, peptide mapping is still used for the same outcomes and has a similar workflow, but technological enhancements have made the process much quicker and more reliable. Protein digestion has benefited from the introduction of bespoke digestion kits that offer highly reproducible digestions in just one hour.
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The introduction of UHPLC and smaller column particles has increased separation speed, and with today’s modern instruments the retention time precision is so high that peptide mapping of known proteins with UV is now possible. Similarly, advancements in mass spectrometry instruments and protein identification software have made identification much more accurate and reliable in a fraction of the previous time, and in line with the increased separation speeds generated by UHPLC.
Peptide mapping is a frequently-performed and necessary analytical technique, and its use is only likely to increase with ever-growing biopharmaceutical pipelines and regulatory guidelines. Over my 15 years of experience with peptide mapping, the workflow has changed very little, but technological advancements have optimised the process to make it much more accurate, reproducible, accessible and fast. Where will peptide mapping be in 2030?
Where would you like to see improvements in the peptide mapping workflow?