Medium Surface Protein Coupling Locus Mass Spectrometry Identification And Application

The coupling sites of protein on the chromatography media are the key factors of controlling of the orientation of the immobilized protein ligands and their bioactivity, which can also affect the separation property of affinity chromatography media. It is important, for improving the separation property of the media, to set up an efficient method to identify the coupling sites of protein ligands on the media and explore a method to control the orientation of the immobilized protein ligands. The present research has used HPLC-MS and enzymatic digestion as the new method to identify the coupling sites of model proteins on the media and also discussed the influence of the activation and coupling processes to the protein coupling sites. This method can also be used to control the coupling sites of protein ligands on media. The present work has given a new angle of the main factors that influence the protein ligands coupling sites on the media and protein activity from the molecule level. The key points are summarized as follows:(1) The research builds up a new method to identify the coupling sites of lysozyme on the media. When Lysozyme was coupled on the Sepharose4FF chromatography media with a22.34μmol/g epoxy group density and coupling reaction time was12h. HPLC-MS and enzymatic digestion were used to identify the coupling sites of lysozyme on the media. It was found that K96, K97and K3were the coupling sites of lysozyme on the media when pH9.5and the proportions of these coupling sites are82%,16%and2%. It is proved that the enzymatic digestion combining the peptide sequence analysis by MS is the efficient method that can be used to identify the protein coupling sites on media.(2) Explored the influence of reaction conditions to the coupling sites of lysozyme on the media. It was found that the epoxy group density and the pH of coupling buffer had a remarkable effect on the coupling sites on the media. When the epoxy group density was low. lysozyme could couple by a single site on the media, and as the increase of the epoxy group density, the number of coupling sites would also increase which could make it difficult to control the site-selective protein immobilization on the media. The pH of coupling buffer had influence on the secondary structure of lysozyme or even caused the aggregation of protein molecules, so a high pH could make the lysozyme coupling on the media by numbers of sites. The coupling pH had an effect on the activity of immobilized Iysozyme on the media. When the epoxy group density was11.34μmol/g and coupled in pH9.5. lysozyme was coupled by a single site which was near the enzyme substrate-binding site thus leading to a low enzyme activity. However, changing the coupling pH to a higher or lower value could make the lysozyme couple on the media by more dispersive amino acid sites, and increase the immobilized lysozyme activity. The coupling time had an important effect on the quantity of the coupled protein ligands on the media, but a less effect on the types of the coupling sites. And it was found that when the coupling time was12h, the quantity of lysozyme ligands could reach the maximum.(3) BSA and CA were used as the model protein to couple on the media with a epoxy group density of22.34μmol/g in pH11.0. The previous method was used to identify the coupling sites. The BSA ligands were mainly coupling on the media by the following amino acids:K180、K114、K20、K294、K474、K375、K173、K159、 K312、K41. and the mainly coupling sites of Carbonic Anhydrase were found to be K17K223and K75HPLC-MS and enzymatic digestion can be used to identify the coupling sites of protein on the media. The reaction condition has an enormous influence on the coupling sites especially the epoxy group density and the pH of the coupling buffer, which can be used to control the coupling sites of protein ligands on the media. The hydrophilicity or hydrophobicity of model proteins was calculated and the result indicated that the hydrophilicity or hydrophobicity of peptides on the surface of the proteins was the critical effect of protein ligands adsorption and coupling on the epoxy media.