A good understanding of this process will help to ensure the maximum sequence coverage and sensitivity are achieved. To first stage in peptide mapping is to break the protein down into peptide fragments by proteolytic enzymes. Like dialysis, this can be used for desalting or for buffer exchange, where the sample buffer is replaced with a new buffer. This is a non-adsorptive chromatography technique that separates molecules based on molecular size. A more robust and well-defined method for desalting samples prior to digestion is Gel Filtration (GF). Although effective, this is a slow process and, since various factors can affect the equilibration process, the final salt concentration can be somewhat variable. Once the equilibration process is complete the sample can be retrieved from the bag. Porosity is chosen so that large molecules, such as the protein of interest, cannot diffuse through the membrane and remain in the bag. The bag is placed in a bag of water or buffer where the concentration of salt will equilibrate through diffusion. It requires filling a dialysis bag (or sometimes a cassette), which is a membrane casing of a defined porosity, with your sample. Dialysis is an established procedure for reducing the salt concentration in samples and can also be used for buffer exchange and small molecule removal. Since peptide mapping is routinely done by LC/MS, desalting is necessary to reduce salts (especially sodium and phosphate) to avoid interference with detection. Each protein will have its own clean-up protocol, but dialysis and desalting are typically required for most protein samples to ensure they are compatible and optimized for digestion. It might be necessary to enrich the sample or to separate the protein from added substances used in the formulation of the product, since these may interfere with either the actual protein digestion or the subsequent LC or LC/MS. The pre-treatment required for your sample will depend on the size and confirmation of your protein 2. The objective of this article is to discuss some of the areas that are important for generating peptide maps by RPC, and highlight considerations for optimising separations to achieve the best possible sequence coverage. Knowledge of sample preparation methods along with efficient separation methods and protocols is required when developing a new peptide mapping strategy 2 A typical peptide mapping profile may include over 100 peaks that each represent an individual peptide and their derivatives. In this case, hydrophilic interaction liquid chromatography (HILIC) may be needed to get sufficient separation to allow identification of the peptide fragments. One example of this is when working with small polar peptides that are not well retained by RPC. 2 Although it is the technique we will focus on here it is worth remembering that, depending on the protein being studied and the experimental objectives, other techniques may also be necessary. The unrivalled resolving power of reversed phase chromatography (RPC) has made it the predominant HPLC technique for separating out peptide fragments. 2įigure 1 illustrates the 62 resulting Herceptin (trastuzumab) peptides following trypsin digestion 20 from the light chains (Lc) and 42 from the heavy chains (Hc). 3 It is important that as much of the protein sequence is covered as possible by the peptide map, since this enables confident protein identification and can provide additional information beyond that obtained at the intact protein level. 2 Figure 1 shows an example of the peptide fragments that are created when Herceptin (trastuzumab) is digested with the enzyme Trypsin, which cleaves following a lysine or arginine amino acids in the protein. The enzyme (usually trypsin) cuts the protein up into peptide fragments at specific points in the protein sequence to produce a “fingerprint” of fragments, which are then chromatographically separated and identified by MS detection. Peptide mapping usually involves enzymatic digestion of a protein. 1 Such modifications may arise during production, processing, and storage and since they may affect the efficacy of a biotherapeutic, they must be thoroughly characterised and controlled. Unlike intact protein analysis, peptide mapping has the advantage of being able to provide side-specific information on post-translational and chemical modifications. 1 It is used to help confirm the identity of a protein therapeutic and to monitor degradations such as oxidation or deamidation. Peptide mapping is a widely used technique to examine the primary structure of biopharmaceuticals.
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