First described in the early 1990s, peptide mapping has emerged as a key technique in many areas of protein research and analysis. Initially part of the proteomics revolution – where researchers looked to break down proteins into their component parts for subsequent identification by mass spectrometry (MS) – peptide mapping is still used today for the initial proof of structure.
Importantly, its application has widened in recent years. Protein mapping is now relied on further downstream in clinical research, during production, and QC. Here, there is a need to not only confirm the sequence of the manufactured protein, but also to ensure that no changes – either single amino acid changes or post-translational modifications (PTMs) – have occurred compared to the original sequence. In addition, it is employed for lot-to-lot identity testing in support of bioprocess development and clinical trials, as well as for monitoring the genetic stability of recombinant cell lines. The technique is also important for developers of biosimilars as a tool for making the essential comparisons with the original drug.
Innovation impacts workflow
The schematic shows a typical protein mapping workflow. While the basic steps have not changed since the inception of the technique, innovation at every stage has improved the technique. As a result, it is now an automated, routine procedure.
A typical peptide mapping workflow
There are two methods for peptide detection that form part of this overall workflow. To confirm the sequence of a known protein, you can use a UV detector and match the peptides found to the control to confirm the sequence. It is a good method of choice if you are confident in your LC instrument, and have a control sample to compare to.
For identification and investigation of PTMs of unknown proteins, you need to detect them on a mass spectrometer. Using this method, you will get the accurate mass of the peptides. These identified peptides can then be matched against a database of known peptide masses, using software to identify the peptides and subsequent protein.
However, challenges remain, and correct LC column choice is critical, particularly for enhanced separation of PTM-containing peptides. Here are some considerations and options.
General considerations for column selection
The inherent complexity of proteolytic digests of protein mixtures means that high-efficiency and high-resolution peptide separation is important. We can see that several factors have an impact:
- Separations performance of the column material – pore size, particle size, and particle morphology (totally porous versus superficially porous) are related to separations efficiency. An ideal column would provide the separations performance required for confident mapping.
- Matching detection technology and mobile phase – current LC/MS methods with MS-friendly formic acid (FA) ion-pairing agent provide increased signal intensity compared to other modifiers. TFA is traditionally used for UV detection. However, the drawback with FA is broader, tailing peaks with many C18 stationary phases leading to coelution of peptide pairs. An ideal column would give sharp peaks with FA.
- Sample – a range of peptides, from hydrophilic to hydrophobic will be in the digest, in addition, larger polypeptides may be present. An ideal column would perform across the wide chemical diversity of peptides.
- Column dimensions – longer columns are required for more complicated maps, and MS sensitivity is related to column diameter. Longer, narrower columns that still yield moderate backpressure are needed. An ideal column range would support this need.
A proven top choice
With these aspects in mind, we can evaluate the new Agilent AdvanceBio Peptide Plus columns. These columns are designed for confident and comprehensive peptide mapping analysis. The HPH Poroshell particles are coated with a modified surface that confers positive charge. The resulting charged surface on this superficially porous particle with C18 ligands improves the peak shape and capacity. AdvanceBio Peptide Plus columns have the optimum pore size (120 Å) and particle size (2.7 µm) to deliver UHPLC-like performance with low column backpressure. These columns provide superior peak shapes with mobile phases containing FA, making them ideal for coupling with mass spectrometry.
A recent application note shows the excellent performance of AdvanceBio Peptide Plus column for monoclonal antibody (mAb) peptide mapping separation with a mobile phase containing FA.
It was evident that AdvanceBio Peptide Plus columns provided sharp and narrow peaks. Within the gradient time, all the peptide peaks were well resolved. The high-resolution and high-efficiency separation achieved provided a peptide map with > 99 % sequence coverage. Precise characterization of PTMs was also achieved.
This kind of superior LC/MS performance is giving users the confidence they need for robust and reliable peptide mapping.
Expert support to add knowledge, and value
Got questions about column choice for peptide mapping? Agilent experts can help. Tune-in to the recording of our recent webinar ‘Choosing the Correct Column and Mobile Phase for LC/MS Peptide Mapping’.
In addition, detailed expert guidance can be found in our ‘How to’ guide, download it here.
Agilent is committed to developing end-to-end workflow analysis solutions for peptide mapping, discover more here.