To date, plenty of new alternative materials for phosphopeptides enrichment prior to mass spectrometry (MS) analysis appear, especially immobilized metal ion affinity chromatography (IMAC) materials. The variable combinations with different metal ions , chelating ligands and solid supports offer full of optionality for IMAC. However, further improvement was predicted by the tedious and complex synthetic process. In this work, a novel covalent organic framework (COF)-based IMAC material (denoted TpPa-2-Ti 4+ ) was prepared simply by direct immobilizing Ti (IV) into TpPa-2 COFs without any extra chelating ligands. The structure and composition of as-prepared composites were confirmed by PXRD, FT-IR and XPS, and a new flower-shaped Ti 4+ -IMAC with regular micro-nano hierarchical structure was observed in the SEM and TEM images. The obtained titanium (IV) ion-modified covalent organic frameworks demonstrated low limit of detection (4 fmol) and largely-satisfactory selectivity (β-casein: BSA=1:100) for phosphopeptide capturing from β-casein. Similarly, 18 and 17 phosphopeptides could be easily detected in the tryptic digest of α-casein or the digest mixture of α-casein and BSA (1:50). They were also successfully applied for enrichment of phosphopeptides from non-fat milk and HeLa cells with high sensitivity and satisfactory selectivity. All above results showed that the new titanium (IV) ion-modified covalent organic framework is expected to be a potential IMAC for phosphopeptide enrichment in large-scale phosphoproteomics studies.
Haiyang Zhang,Xiaowei Li,Yating Yao,Shujuan Ma,Zhen Liu,Junjie Ou,Yinmao Wei,Mingliang Ye
Hierarchically porous monoliths as a key feature of biological materials have been applied in enrichment and separation. In this work, a metal immobilized hierarchically porous organosilica hybrid monolith was synthesized by hydrolysis and condensation of tetraethoxysilane (TEOS) and diethoxyphosphorylethyl-triethoxysilane (DPTS) under alkaline environment . Phosphonate ester groups were firstly introduced by the employment of DPTS as functional monomer , and then acidified to phosphonic acids . The surface area of optimal monolith could reach to 1170 m 2 /g, which simultaneously contained micropores and mesopores (4 nm) obtained from nitrogen sorption measurement. Meanwhile, mercury intrusion porosimetry (MIP) further demonstrated that macropores (1–3 μm) existed in monoliths. Followed by chelating with titanium ion (Ti 4+ ), the hierarchically porous organosilica hybrid monoliths could be applied as IMAC materials. This synthesized process was easy-operating and time-saving, and avoided the tedious and complex process of traditional Ti 4+ -IMAC materials. Furthermore, the Ti 4+ -IMAC monoliths exhibited high adsorption capacity for pyridoxal 5′-phosphate (82.6 mg/g). The 3282 unique phosphopeptides could be identified from 100 μg of HeLa digests after enrichment with the monolith, exhibiting excellent enrichment performance of low-abundance phosphopeptides.
Lei Pan,Shujuan Ma,Ruizhi Tang,Wenrui Wu,Junjie Ou,Cong Li,Yehua Shen
Protein phosphorylation is involved in many biological processes and associated with some diseases. However, because of the low abundance and large dynamic changes of phosphopeptides in biological samples, it is necessary to enrich phosphopeptides before mass spectrometry detection. Many kinds of materials have been developed for phosphopeptide enrichment, but most of them are disposable and their preparation processes are toxic. In this study, a green strategy was presented to fabricate immobilized metal affinity chromatography (IMAC) materials. Using chitosan as a precursor, polyethylene glycol diglycidyl ether as a cross-linking agent and 2,3,4-trihydroxybenzaldehyde as a functional monomer, a series of chitosan membranes (CMs) with free-standing lamellar 3D architectures were prepared via a freeze-casting technique and after chelating with titanium ions (Ti4+) could serve as IMAC adsorbents for phosphopeptide enrichment in biological samples. Due to the stronger chelating force between Ti4+ and the pyrogallol ligand than that between Ti4+ and the phosphate groups, the IMAC material is reusable for the enrichment of phosphopeptides, exhibiting an excellent enrichment performance and a higher capacity than other IMAC materials such as organosilica hybrid monolith and carbonaceous spheres. Besides, four endogenous phosphopeptides and two dephosphorylated fragments were captured by Ti4+-CM from the human serum. Therefore, Ti4+-CM demonstrates potential commercialization prospects because of its green preparation process and reusability.
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