Yiren Cao,Youli Tian,Ke-Er Chen,Yixin Ma,Qiang Zhang,Weiwen Liu,Zhishen Mu,Shengpeng Su,Chengxi Cao
Herein, we report a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method featuring dual imaging and signal-fusion deep learning for specific identification and analysis of glycomacropeptide (GMP) in milk sample. Conventional SDS-PAGE methods lack specificity because of the signle staining of protein bands, and the overlap between GMP and β-lactoglobulin (βLg). Our dual imaging method generated a pair of complementary detection signals by recruiting intrinsic fluorescence imaging (IFI) and silver staining. Comparing the IFI image with the staining image highlighted the presence of GMP and differentiated it from βLg. Additionally, we trained a signal-fusion deep learning model to improve the quantitative performance of our method. The model fused the features extracted from the paired detection signals (IFI and staining) and accurately classified them into different mixing ratios (proportion of GMP-containing whey in the sample), indicating the potential for quantitative analysis on the mixing ratios of GMP added into whey sample. The developed method has the merits of specificity, sensitivity and simplilcity, and has potential to analysis of protein/peptides with unique IFI properties in food safety, basic research and biopharming etc.
Yu Lu,Jie Liu,Zhenhua Li,Wenqing Li,Jing Liu,Linjuan Huang,Zhongfu Wang
Casein glycomacropeptide carries various O-glycan modifications, which, together with variations in the amino acid composition of the glycopeptide, may result in different biological activities. In this study, O-glycans of casein glycomacropeptide from bovine and caprine whey powder were qualitatively and quantitatively analyzed by LC-UV-ESI-MS/MS, and their immune activities and regulatory mechanisms were compared. O-Glycans’ total content was 1.54 times higher in bovine than in caprine glycomacropeptide. The glycoform H1N1S2 (H: hexose; N: N-acetylgalactosamine; and S: N-acetylneuraminic acid) accounted for nearly 50% of total glycomacropeptide O-glycans in bovine milk but less than 20% in caprine milk. Bovine glycomacropeptide glycosylation promoted the immune activity of RAW264.7 cells, which may be linked to a higher content of disialylated O-glycans. Glycomacropeptide from both milk sources significantly upregulated the mRNA expression of IL-1α, TNF-α, and IL-10 in RAW264.7 cells and activated the MAPK immunomodulatory signaling pathway. This study demonstrates the possible use of casein glycomacropeptide as an immunomodulatory agent.
Glycomacropeptide (GMP) is a bioactive peptide of high value, rich in glycosylation sites and with physiological and dietary therapeutic value. The enrichment and detection of GMP facilitates the accurate quantification and the identification of adulteration of GMP in food products. In GMP, sialic acid is an abundant glycosyl group and is mainly located at the end of the sugar chain. Here, we propose a novel GMP enrichment strategy based on the affinity of sialic acid for phenylboronic acid groups that shift with environmental pH. As an enrichment material, mesoporous silica nanoparticles were progressively modified with aminopropyl and phenylboronic acid groups. The developed material showed excellent selectivity for sialic acid in the presence of galactose and fucose as interferents. The adsorption behavior of sialic acid-containing GMP fits the Langmuir adsorption model, offering a recovery of 71.72% (in terms of sialic acid content) and a GMP relative purity of 0.957. Results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography confirm that the enriched GMP contains almost no other unexpected proteins and peptides, indicating that the developed strategy holds promise for purifying GMP in various dairy systems.
危险品化学品经营许可证（带存储）