Introduction

Exosomes, widely distributed in body fluids such as blood and urine, are secreted by various living cells and play an irreplaceable role in intercellular molecular transport and signal transduction [1,2,3,4,5]. Studies have shown that exosomes are associated with pathological changes in some diseases and may become related biomarkers [6,7,8]. Phosphorylation is a fundamental and significant protein post-translational modification [9], involved in various processes such as cell metabolism, cell apoptosis, energy conversion, and signal transduction [10, 11]. Some studies have demonstrated that phosphorylation mediates the occurrence and development of some diseases by participating in exosome biogenesis [12]. Exosomal phosphorylated proteins may be potential biomarkers to achieve early diagnosis of diseases [13]. Therefore, the analysis of exosomal phosphopeptides is beneficial to elucidate the pathogenesis of related diseases.

Currently, the immobilized metal ion affinity chromatography (IMAC) strategy is one of the most popular strategies for phosphopeptide enrichment [14, 15]. Numerous substrates have been developed to fix metal ions, such as graphene, mesoporous silica, and metal/covalent organic frameworks [16,17,18]. However, the most prepared IMAC materials not only need complex steps but also have finite binding sites for metal ions due to the limitations of the substance themselves. In addition, for most substrates used to prepare IMAC materials, due to the limited hydrophilicity and small specific surface area, it is necessary to select suitable organic ligands to construct IMAC materials with high hydrophilicity and large specific surface area. Covalent organic frameworks (COFs) have attracted extensive interest due to the simple synthesis procedure, good chemical stability, and abundant functional groups [19, 20]. Among the many types of organic structural units, β-CD is a very promising ligand for constructing COF due to its rich groups. By braiding β-CD into a framework through reticulation chemistry, the resulting β-CD-COF has high chemical stability [21], which has greatly broadened its applications in proteomics. Therefore, β-CD-COF carries abundant chelation sites favorable for the chelation of more metal ions and has the potential to be an effective IMAC-based nanomaterial to achieve the enrichment of phosphorylated peptides and exosomes.

Herein, a Ti4+ functionalized β-cyclodextrin covalent organic framework was developed via a new one-pot method and used as an IMAC platform for the enrichment and separation of phosphorylated peptides and exosomes. We prepared β-CD-COF by using 2,3,4-trihydroxybenzaldehyde (THBA), p-phthalaldehyde (TPA), and Heptakis-(6-amino-6-deoxy)-cyclodextrin (Am7CD) via Schiff base reaction under mild reaction conditions. Subsequently, by means of the abundant cis dihydroxyl groups carried by the organic ligand THBA introduced in the prefabricated framework, a large number of Ti4+ was firmly fixed onto the β-CD-COF substrate, thus endowing the proposed material with adsorption capacity for phosphopeptides. Furthermore, benefiting from the novel organic ligand structure and the introduction of metal ions, the functionalized β-CD-COF@Ti4+ provides a new pathway to detect and separate phosphorylated peptides and exosomes.

Experimental section

Chemicals

p-Phthalaldehyde (TPA), 2,3,4-trihydroxybenzaldehyde (THBA), 2,5-dihydroxybenzoic acid (DHB), acetic acid (HAc), and phosphate buffer solution (PBS) were purchased from Macklin. Trypsin, albumin from bovine serum (BSA), ethanol (EtOH), acetonitrile (ACN), and β-casein (98%) were bought from Sigma-Aldrich. Heptakis-(6-amino-6-deoxy)-β-cyclodextrin (Am7CD), trifluoroacetic acid (TFA), titanic sulfate, iodoacetamide (IAA), and dithiothreitol (DTT) were bought from Aladdin. The serums of patients with uremia were obtained from the Affiliated Hospital of Medical School, Ningbo University, with the approval from its Ethics Committee (KS20227002).

Synthesis of β-CD-COF

A total of 114.4 mg of Am7CD, 87 mg of TPA, and 78.2 mg of THBA were dispersed ultrasonically in a centrifuge tube (10 mL) containing ethanol (2.5 mL) and water (2.5 mL), followed by the addition of 0.05 mL of HAc (176 mM). After stewing at room temperature for 50 h, the resulted β-CD-COF was collected and thoroughly washed with water and EtOH 3 times, respectively. The β-CD-COF was dried under vacuum for 24 h at 50 °C.

Synthesis of β-CD-COF@Ti4+

Firstly, 40 mg of β-CD-COF was added into 12 mL of titanium sulfate solution (100 mM) and oscillated for 4 h at room temperature. Then, the synthesized β-CD-COF@Ti4+ was dried under vacuum.

β-CD-COF@Ti4+ was synthesized by a simple two-step procedure (Fig. 1). First of all, Am7CD, THBA, and TPA were added into the mixed solution of water and ethanol, and the imide condensation reaction was carried out under the catalysis of acid to obtain β-COF. Then, the solid material was immersed in a certain concentration of the solution of titanic sulfate, and the Ti4+ was chelated with the hydroxyl group in THBA to acquire the Ti4+ functionalized β-cyclodextrin covalent organic framework (β-CD-COF@Ti4+).

Fig. 1
figure 1

The schematic representation of the preparation process of β-CD-COF@Ti4+ composites

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Enrichment of phosphopeptides from standard protein digests

β-CD-COF@Ti4+ (100 μg) was added into the loading buffer (50% ACN, 0.5% TFA) containing β-casein digest (2 μL). The material loaded with the phosphopeptides was washed with loading buffer several times after vibrating at 37 °C for 45 min. Then, 10 μL of eluent (0.4 mol/L NH3∙H2O) was poured, and the solution was shaken for 30 min at 37 °C. The eluent was obtained and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Enrichment of phosphopeptides from biological samples

β-CD-COF@Ti4+ (1 mg) was put into the loading buffer (100 μL) including 2 μL of human serum. The mixture was washed five rounds with loading buffer after vibrating at 37 °C for 45 min. Next, 2 × 10 μL of eluent was incubated at 37 °C for 0.5 h. Then, the eluent was lyophilized and desalted for further experiments. The pretreatment process of serum could be seen in supporting information.

Except for the practical biological samples (20 μL), the steps for enriching phosphopeptides from defatted milk were the same as the above experiments for enriching phosphopeptides from human serum. The pretreatment of defatted milk could be seen in supporting information.

Enrichment of the exosomes from human serum

β-CD-COF@Ti4+ (3 mg) was added into the centrifuge tube containing 100 μL of human serum and vibrated for 45 min at 4 °C. The pretreatment of serum could be seen in supporting information. Then, the material was washed with low-temperature PBS for 3 times. Subsequently, the eluent (10 μL) was put in a centrifuge tube and vibrated at 4 °C for 0.5 h. Lastly, the mixture was obtained and used for follow-up experiments.

Results and discussion

The optimization of synthesis conditions and characterization of β-CD-COF@Ti4+

A range of characterization experiments were performed to verify the synthesis of the β-CD-COF@Ti4+. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of β-CD-COF and β-CD-COF@Ti4+ showed that the materials were nanospheres with a diameter of about 500 nm (Fig. S1 and Fig. 2). The energy dispersive X-ray spectra (EDX) demonstrated the presence of Ti4+ (Fig. S2). Besides, Fourier-transform infrared spectroscopy (FT-IR) was carried out (Fig. S3). The peaks at 1693 cm−1, 1769 cm−1, and 1785 cm−1 assigned to the vibration of C=O disappeared, and the peaks at 1597 cm−1 and 3322 cm−1 attributed to the vibration of C=N appeared [22,23,24], indicating the success of the Schiff base reaction.

Fig. 2
figure 2

a SEM image of β-CD-COF@Ti4+; b TEM image of β-CD-COF@Ti4+

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To optimize the synthesis of β-CD-COF, the materials were prepared with different molar ratios (β-CD: THBA = 1:2, 1:5, 1:10). According to the best experimental results in Fig. S4, the ligand ratio was determined to be 1:5 in subsequent experiments. The analytical process for the optimization of the ratio of β-CD-COF materials can be obtained from supporting information.

Application of β-CD-COF@Ti4+ for the enrichment of phosphopeptides from standard protein digests

Trypsin digested β-casein was employed as an enrichment sample to verify the enrichment performance of β-CD-COF@Ti4+. In order to obtain the best enrichment ability for phosphopeptides, the optimal incubation time (15 min, 30 min, 45 min, and 60 min) was studied. MALDI-TOF MS analysis results indicated that the optimal incubation time was 45 min (Fig. S5). To investigate the mechanism of the materials for separation and identification of phosphorylated peptides, β-CD-COF was used to enrich phosphopeptides under the same conditions. Before enrichment (Fig. S6a), non-phosphorylated peptide peaks dominated the spectrum. After enrichment by β-CD-COF, only 1 phosphopeptide could be observed (Fig. S6b). Besides, the background baseline of the mass spectrum was relatively high. Then, after enrichment by β-CD-COF@Ti4+, 13 phosphopeptides could be observed (Fig. S6c), and the detailed information about phosphorylated peptides was listed in Table S1. The result indicated that the mechanism of the materials for separation and identification of phosphorylated peptides was mainly based on the coordination of Ti4+ and phosphate groups.

The β-casein digests were diluted to different concentrations (500, 50, 5, and 0.5 fmol) to explore the detection limit of β-CD-COF@Ti4+ towards phosphopeptides. After diluting the β-casein concentration to 500 fmol, 6 phosphopeptides with S/N > 3 were captured by β-CD-COF@Ti4+ (Fig. S7a). Five phosphopeptide peaks with S/N > 3 could be observed when diluting the β-casein digests to 50 fmol (Fig. S7b). Two phosphopeptide peaks (Fig. S7c) with S/N > 3 were also seen at a concentration of 5 fmol. Additionally, even the concentration down to 0.5 fmol, 1 phosphopeptide peak (m/z = 2062.8, S/N > 3) could be observed (Fig. S7d). Based on the above analysis, β-CD-COF@Ti4+ showed low detection for phosphopeptides.

BSA digests were used as the interfering peptides to verify the selectivity of β-CD-COF@Ti4+ towards phosphopeptides. When the BSA/β-casein at molar ratio was 100:1, 7 phosphopeptide peaks dominated the spectrum (Fig. S8a). When the molar ratio was 250:1, 5 phosphopeptide peaks were observed (Fig. S8b). Three phosphopeptide peaks (Fig. S8c) were observed with the molar ratio that was 500:1. A single phosphopeptide peak was still identified at a molar ratio of 1000:1 (Fig. S8d). These results demonstrated the good selectivity of β-CD-COF@Ti4+.

The peak intensity changes of three typical phosphopeptides (m/z = 2062, 2556, 3123) were used to assess the phosphopeptide loading capacity of β-CD-COF@Ti4+. Each experiment was performed 3 times, and the RSDs were given in Table S2. The experiment was performed by changing the amount of β-CD-COF@Ti4+ and fixing the quantity of β-casein digests (10 μg). With an increasing amount of β-CD-COF@Ti4+, the intensity of the peaks also increased (Fig. S9). The peak intensity was almost stable when the mass of β-CD-COF@Ti4+ was 100 μg, and the peak intensity was almost stable when the amount of material further increased. The loading capacity of β-CD-COF@Ti4+ was approximately 100 mg·g−1 by calculating the formula Q =m1/m2 (m1 represents the amount of β-casein digests, and m2 represents the amount of material).

A comparison of the relevant properties of our work with recently reported IMAC nanomaterials is given in Table S3. Benefiting from the selection of organic ligands, β-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, such as a low detection limit, high sensitivity, and high loading capacity. Am7CD could be grafted with more organic ligands (THBA, TPA) due to its abundant amino groups; the structural unit THBA introduced in the β-CD-COF framework had multiple hydroxyl affinity sites, which provided a platform for the subsequent binding of more metal ions (Ti4+) and improved the hydrophilicity of material. In addition, compared with Fe3O4@mPDA@Ti4+, β-CD-COF@Ti4+ had a higher loading capacity due to its large specific area. Excitingly, there were few reports on the application of Ti4+ functionalized β-cyclodextrin covalent organic frameworks in proteomics. Therefore, the effective building of β-CD-COF@Ti4+ provided a feasible route for the construction of functionalized β-cyclodextrin COF and a platform for the enrichment of phosphopeptides.

Application of β-CD-COF@Ti4+ for the enrichment of phosphopeptides from complex bio-samples

Defatted milk is a classic biological sample containing phosphoproteins. Based on the above results, β-CD-COF@Ti4+ was further used to detect phosphopeptides in defatted milk. Before treatment with β-CD-COF@Ti4+, no phosphopeptides were observed, and non-phosphopeptides dominated the spectrum (Fig. S10a). After treatment with β-CD-COF@Ti4+, nine phosphopeptides of high mass intensity and a clear background were observed (Fig. S10b). Detailed information was presented in Table S4. In summary, the results of the study on complex samples provided strong proof for the good performance of the obtained materials on the capture of phosphopeptides.

Serum is a typical clinical specimen that contains a large number of substances necessary for life activities, such as endogenous peptides and proteins. From the current research status and disease characteristics, the discovery of specific proteins related to disease lesions could be used to assist in the diagnosis of diseases, and it was important for the elaboration of relevant diseases. After treatment with β-CD-COF@Ti4+, 29 phosphopeptides related to 23 phosphoproteins from the normal control group and 24 phosphopeptides related to 22 phosphoproteins in the uremia patient group were detected (Fig. S11), respectively. As shown in Fig. 3a, a total of 23 phosphorylation sites, 21 phosphopeptides, and 18 phosphoproteins were only detected in normal samples. By comparison, a total of 20 phosphorylation sites, 17 phosphopeptides, and 16 phosphoproteins were detected only in uremia samples. The relevant information was in supporting information Table S5 and S6.

Fig. 3
figure 3

a Venn diagram of phosphopeptides, phosphoproteins, and phosphorylation sites of normal control and uremia patient; b the sequence logo of identified phosphopeptides from normal control and uremia patient

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To assess the significant differences between healthy controls and uremia patients, we centered on the phosphorylation sites and visualized the amino acid frequencies through WebLogo. By comparing the results of normal and uremic patients (Fig. 3b), we found no significant differences in the phosphorylation sites among the three main amino acids (tyrosine, threonine, and serine). Notably, the possibility of valine, leucine, and isoleucine near the phosphorylation sites was greatly increased in patients with uremia, and the changes in these metabolites may be related to the pathogenesis of uremia, which may have actual significance for the clinical diagnosis and treatment of uremia [25].

Besides, the terms of Gene Ontology (GO) analysis were conducted to distinguish the phosphoproteins of the two samples through the DAVID database v6.8, in an attempt to explore valuable biological information from the perspectives of molecular function, cellular component, and biological process (Fig. 4). From the molecular function, uremia patients were often accompanied by altered lipoprotein metabolic processes. Abnormal lipoprotein metabolism was common in uremia and significantly affected the development of kidney-related diseases. [26, 27]. As can be seen from the cellular component, very-low-density lipoprotein (VLDL) particle and high-density lipoprotein (HDL) particle are highly expressed in the serum of uremia patients. According to previous reports, the VLDL particles in dialysis patients contain more cholesterol and phospholipids than healthy controls [28]. In addition, the high-density lipoprotein (HDL) particles of uremic patients have changed, which may lead to their loss of vascular protection function and conversion into inflammatory particles, leading to further development of the disease [29, 30]. From the biological process perspective, lipid binding is thought to be closely associated with phosphoproteins in the serum of uremic patients. It has been reported that renal dysfunction in uremia patients may affect lipid binding activity, thereby affecting the entire lipid metabolism process [31]. In summary, the pathological process of uremia may involve these functional pathways, which is instructive for relevant diagnosis and treatment.

Fig. 4
figure 4

The GO analysis of phosphoproteins identified in the serum of normal control (a) and uremia patient (b)

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Application of β-CD-COF@Ti4+ for the enrichment of exosomes from serum

We further applied β-CD-COF@Ti4+ in the enrichment of exosomes. As shown in the TEM image (Fig. 5a), the morphology of exosomes captured by β-CD-COF@Ti4+ was spherical in structure with a diameter of about 30–150 nm, in good agreement with the previous articles [32,33,34]. To investigate the mechanism of the materials for separation and identification of exosomes, β-CD-COF was used to enrich exosomes under the same conditions. Besides, western blot further validated the promising application of two materials (β-CD-COF and β-CD-COF@Ti4+) in the isolation and enrichment of exosomes. After enrichment by β-CD-COF, three marker proteins (CD9, CD63, TSG101) of exosomes were barely observed (Fig. 5b). By contrast, after enrichment by β-CD-COF@Ti4+, three marker proteins (CD9, CD63, TSG101) of exosomes were detected (Fig. 5c) [35]. β-CD-COF@Ti4+ could capture exosomes through the adsorption of metal ions towards the phospholipid layer, based on the IMAC strategy. In other words, β-CD-COF@Ti4+ has great potential for applications in the capture and separation of exosomes.

Fig. 5
figure 5

a TEM image of exosomes captured by β-CD-COF@Ti4+; western blot results of marker proteins in exosomes after enrichment by b β-CD-COF and c β-CD-COF@Ti4+

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Conclusions

All in all, we developed a β-cyclodextrin covalent organic framework by a new synthetic process. The presence of sufficient cis dihydroxyl groups on the β-CD-COF layer allowed for more chelation of Ti4+, providing a better platform based on the IMAC strategy for the separation of phosphorylated peptides and exosomes from biological samples. β-CD-COF@Ti4+ showed excellent selectivity, low detection limit, and good ability towards phosphopeptides. The limitation of this strategy is the use of organic reagents, so exploration of an environmental-friendly synthesis method is needed in the future phosphoproteomics research.