Facile synthesis of titanium-rich carbonaceous material via Schiff base reaction for efficient separation and analysis of phosphorylated peptides and exosomes from human serum

Abstract

Efficient capture of phosphorylated peptides and phosphorylated exosomes is important for in-depth proteomics research. In this work, a carbonaceous material (denoted as CTs@DHA) was synthesized based on the Schiff base reaction by hydrothermal carbonization using chitosan (denoted as CTs) and 2,3-dihydroxy terephthalaldehyde (denoted as DHA) as ligands and water as the sole component of the solvent. The most important thing is that the experimental process is simple, environmentally friendly, and time-saving. Based on the immobilized metal affinity chromatography (IMAC) strategy, titanium ions were chelated with CTs@DHA to obtain a novel material (denoted as CTs@DHA@Ti4+) to capture exosomes and phosphopeptides. The CTs@DHA@Ti4+ captured phosphopeptides with high selectivity (the molar ratio of BSA to β-casein was 2000:1) and satisfactory sensitivity (2 fmol), with excellent loading capacity (83.3 μg/mg). Additionally, this material was successfully applied in complex bio-sample analysis; four endogenous phosphopeptides from the serum of uremic patients, 16 phosphopeptides from human saliva, and exosomes from human serum were detected. After being analyzed with nano-LC MS/MS, 12 phosphopeptides with 10 phosphorylation sites from 10 phosphoproteins were detected in normal serum, and 14 phosphopeptides with 16 phosphorylation sites from 13 phosphoproteins were detected in the serum of uremic patients. All experiments have shown that this material provides an effective method for isolating exosomes and enriching phosphopeptides, and it has bright application prospects in disease research and proteomics research.