Design and synthesis of affinity ligands and relation of their structure with adsorption of proteins.

Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research.

Pre-fractionation of rat liver cytosol proteins prior to mass spectrometry-based proteomic analysis using tandem biomimetic affinity chromatography.

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low-copy-number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre-fractionation were digested and then analyzed by LC-MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un-fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un-fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre-fractionation with MS-based proteomic analysis is simple, low-cost, and effective, providing the prospect of broad application in proteomics.