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.

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Edible insects are alternative sources of high protein. Southeast Asia, South America, Africa, and Europe have recorded edible insects and, especially in Europe and America, there are factories that produce insects as food. In some areas of China, there has also been the habit of eating insects since ancient times. Periplaneta americana is an insect with the homology of medicine and food. In recent years, its medicinal and nutritional functions have attracted extensive attention and research. Its adult powder has been certified as a health product. It not only contains a variety of proteins but also is rich in fatty acids. The composition and antioxidant function of adult powder extract was analyzed in this study. Using adult P. americana powder as the raw material, it was extracted with n-Hexane, dichloromethane, and ethyl acetate, respectively. Gas chromatography-mass spectrometry (GC-MS) removed a total of 60 compounds, and many active components were extracted from P. americana powder for the first time. The parts and relative content of each extracted sample were obtained, and the total antioxidant capacity (T-AOC) of each extracted piece was determined, among which the antioxidant activity of ethyl acetate was the highest.

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.

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