RESUMEN
Chitosan, as a biological macromolecule with excellent biocompatibility, has great potential for application in immobilized metal affinity chromatography (IMAC) strategies. In-depth analysis of low-abundance phosphopeptides in organisms can help reveal the pathological mechanisms of diseases. Here, we developed an IMAC material based on a biomimetic honeycomb chitosan membrane. The material demonstrates excellent biocompatibility, good hydrophilicity, and strong metal chelating capacity, which collectively confer outstanding enrichment properties. The material has high sensitivity (0.05 fmol), great selectivity (1:2000), excellent cycling stability (at least 10 cycles) and acid-base stability. In addition, the material was employed in human serum, successfully enriching 129 phosphopeptides from the serum of gastric cancer patients and 146 phosphopeptides from healthy controls. Sequence logo suggests a potential association between gastric cancer and glutamine. Ultimately, an in-depth gene ontology analysis was carried out on the phosphopeptides that were enriched in the serum samples. Compared to normal controls, our results demonstrated dysregulated expression of biological process, cellular component, and molecular function in gastric cancer patients. This suggests that the disease involves, such as blood coagulation pathways, cholesterol metabolism, and heparin binding. All experimental outcomes converge to demonstrate the substantial promise of the material for applications within proteomics research.
RESUMEN
A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol), high selectivity (ß-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143 mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.