Simultaneous Adsorption of Intact Glycoproteins and Phosphoproteins Using Hydrophilic Titanium (IV) Ion-Immobilized Cotton Fiber Functionalized by Phytic Acid Assembly

Zhang, Dawei; Zhou, Zhenhua; Sun, Guifeng; Chen, Shiying; Jiang, Minzhi; Yao, Zihan; Chukwunonso Ogbuehi, Anthony; Wang, Hao; Jin, Lei

doi:https://doi.org/10.1155/2023/9943919

Journal of Chemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Abbreviations Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 9943919 | https://doi.org/10.1155/2023/9943919

Simultaneous Adsorption of Intact Glycoproteins and Phosphoproteins Using Hydrophilic Titanium (IV) Ion-Immobilized Cotton Fiber Functionalized by Phytic Acid Assembly

Dawei Zhang,¹Zhenhua Zhou,²Guifeng Sun,¹Shiying Chen,³Minzhi Jiang,³Zihan Yao,³Anthony Chukwunonso Ogbuehi,⁴Hao Wang,⁵and Lei Jin³

Academic Editor: Christos Kordulis

Received13 Jan 2023

Revised20 May 2023

Accepted26 May 2023

Published06 Jun 2023

Abstract

Protein glycosylation and phosphorylation are two important post-translational modifications and play significant roles in various biological processes. However, both glycoproteins and phosphoproteins are in low abundance in complex samples, which hinders their characterization. In this work, we developed a novel method for the simultaneous adsorption of glycoproteins and phosphoproteins based on solid phase extraction. By virtue of the unique structure of hydrophilic titanium (IV) ion immobilized cotton fiber functionalized with phytic acid (denoted as Ti⁴⁺-PA-cotton), glycoproteins, and phosphoproteins could be efficiently enriched in one-step incubation and eluted in sequence. The newly prepared Ti⁴⁺-PA-cotton was well characterized, and the adsorption isotherms and kinetics were studied. The maximum adsorption capacities of Ti⁴⁺-PA-cotton for β-casein and HRP were 833.3 mg/g and 384.6 mg/g with the adsorption rate of 259.1 and 63.1 mg/g·min. Standard protein mixture of BSA, HRP, and β-casein was used to test the enrichment ability as a proof of concept. The SDS-PAGE results demonstrated that Ti⁴⁺-PA-cotton had an excellent enrichment performance and possessed an outstanding application potential in the analysis of glycoproteins and phosphoproteins.

1. Introduction

Glycosylation and phosphorylation, two ubiquitous post-translational modifications (PTMs) of proteins, play significant roles in various biological processes. It has been estimated that nearly 50% of proteins are glycosylated and 30% of proteins are phosphorylated in mammals [1–3]. Protein glycosylation and phosphorylation were involved in the numerous biological events, such as gene expression, molecular recognition, signaling transduction, and immune response [4–6]. Aberrant glycosylation has been proven to be related to many human diseases, such as cardiovascular disease, cancer, and congenital disorders [7, 8]. The majority of the currently available FDA-approved tumor biomarkers are glycoproteins. Meanwhile, phosphoprotein dysfunction was also verified to be critically involved in the pathogenesis of cancers [9], Alzheimer’s disease [10], and Parkinson’s disease [11]. For this reason, the study of glycosylation and phosphorylation is of great importance. Nevertheless, both glycoproteins and phosphoproteins were in low abundance in biological samples, and their detection signal was easily suppressed by other non-PTM proteins. Thus, the selective separation and enrichment of glycoproteins and phosphoproteins were prerequisite in the process of sample pretreatment.

Over the last decades, lots of enrichment strategies were proposed for the adsorption of glycoproteins and phosphoproteins. Most of the enrichment strategies for the specific enrichment of glycoproteins and phosphoproteins were mutually independent. For glycoprotein/glycopeptide enrichment, lectin-affinity chromatography [12], boronic acid affinity materials [13], hydrazide chemistry [14], and hydrophilic interaction liquid chromatography (HILIC) [15] were developed and widely used ascribing to the advantages of easy operation and universality. As for phosphoprotein/phosphopeptides enrichment, immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) were the two main enrichment strategies due to their merits of low cost and ease-of-use [16–19].

Although great achievements have been made in the field of glycoproteins/glycopeptides and phosphoproteins/phosphopeptides, most of the developed materials possessed only one function of enriching glycoproteins/glycopeptides or phosphoproteins/phosphopeptides; this kind of strategy would suffer from problems when the amount of biological sample was low and precious. Thus, a strategy for the simultaneous enrichment of phosphorylated and glycosylated proteins was proposed. The main advantage of simultaneous enrichment is the ability to obtain more comprehensive and accurate information on cellular signaling pathways involving synergistic regulation of glycosylation and phosphorylation. Simultaneous enrichment could also promote a deeper understanding of key proteins and their interactions in complex protein networks, help to explore and discover new biological mechanisms and indicators, and improve the diagnosis and treatment of diseases. Recently, several novel materials were designed for the enrichment of both glycopeptides and phosphopeptides. However, some of the materials were used in different enrichment conditions when enriching different targeted peptides [20, 21]; some others were able to enrich glycopeptides and phosphopeptides in a single step but could not achieve the sequential elution [22, 23]. Only a few research studies were reported on the simultaneous enrichment of glycopeptides and phosphopeptides and stepwise elution [24–28], and these research studies focused on the enrichment of the peptides rather than the intact proteins. The top-down strategy in which intact phosphoproteins and glycoproteins are enriched and analyzed directly without enzymatic digestion is a complementary to the bottom-up strategy and could also obtain the full molecular information and cross-talk of different PTMs. Therefore, more research efforts are still needed to develop new materials and integrated approaches for their simultaneous enrichment with high selectivity and specificity by a facile approach.

In this work, we prepared a novel composite material consisting of a cotton fiber functionalized with phytic acid and immobilized titanium (IV) ions (denoted as Ti⁴⁺-PA-cotton) for the simultaneous enrichment of glycoproteins and phosphoproteins in a single step, and then the enriched glycoproteins and phosphoproteins could be eluted sequentially using different elution buffers. To the best of our knowledge, there are few studies aiming at the simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. The Ti⁴⁺-PA-cotton with unique architecture was specifically designed and could be facilely synthesized. Cotton, as a natural raw material, is mainly made up of cellulose (over 90%), which possesses excellent hydrophilicity due to its polar hydroxyl groups. Thus, cotton could capture glycan moieties of glycoproteins/glycopeptides through hydrophilic interaction [29]. Phytic acid (PA) is a natural compound available from plant sources and reported to be readily modified onto the surface of different materials through one-step assembly reaction. The assembly was triggered via various forces mainly dominated by hydrogen bond [30]. The six phosphate groups of PA molecule could provide abundant affinity sites for the immobilization of titanium ions, which were able to coordinate with the phosphate groups of phosphoproteins. The prepared Ti⁴⁺-PA-cotton was nontoxic, biodegradable, biocompatible, and environmentally friendly. In addition, Ti⁴⁺-PA-cotton was fitted into a pipette tip for the solid phase extraction (SPE) of glycoproteins and phosphoproteins. The SPE approach based on Ti⁴⁺-PA-cotton combined HILIC strategy and IMAC strategy, which could reduce enrichment time, facilitate enrichment operation, and was favorable for its application and promotion. The adsorptions of BSA, HRP, and β-casein on Ti⁴⁺-PA-cotton were studied. The enrichment performance of Ti⁴⁺-PA-cotton SPE tip was validated by SDS-PAGE, which demonstrated a promising prospect in scientific research and clinical study of glycoproteins and phosphoproteins.

2. Materials and Methods

2.1. Materials

Acetonitrile (ACN), trifluoroacetic acid (TFA), ammonia hydroxide solution (28% v/v in H₂O), phytic acid (PA, 70% in H₂O), and iron chloride hexahydrate were purchased from Aladdin (Shanghai, China). Peroxidase (HRP) and β-casein were purchased from Sigma Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from X-Y Biotechnology (Shanghai, China). Titanium (IV) sulfate was purchased from Macklin (Shanghai, China). PageRuler™ Prestained Protein Ladder (10 to 180 kDa) was purchased from Thermo Fisher Scientific (Wyman Street, Waltham, MA, USA). NNNN’-tetramethylethylenediamine (TEMED), 30% Acr-Bis, 1.5M Tris-HCl (pH 8.8), 1M Tris-HCl (pH 6.8) and 6 × SDS-PAGE sample loading buffer were purchased from Beyotime Biotechnology (China). Sodium chloride, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate (APS), degrease cotton, BCA protein assay kit, and protein stains H were purchased from Sangon Biotech (Shanghai, China). Human serum was obtained from a healthy volunteer. Ultrapure water (18.2 MΩ·cm) was produced by Millipore Simplicity® system (Billerica, MA, USA). All the other reagents were of analytical grade or better and used without further purification.

2.2. Preparation of Ti⁴⁺-PA-Cotton

The facile synthetic procedure of Ti⁴⁺-PA-cotton involved two main processes, (i) the degrease cotton was functionalized with phytic acid (PA) to prepare PA-cotton, and (ii) titanium cations were immobilized onto the surface of PA-cotton to obtain Ti⁴⁺-PA-cotton. Detailed preparation protocols were described in the supplementary material (SM).

2.3. Characterization of Ti⁴⁺-PA-Cotton

Ti⁴⁺-PA-cotton was characterized by scanning electron microscopy (SEM, FEI Quanta FEG 250) to observe its morphology, and the elemental distribution onto the surface of Ti⁴⁺-PA-cotton was studied by SEM equipped with energy-dispersive spectrometry (EDS) microanalysis unit. The functional groups of Ti⁴⁺-PA-cotton were analyzed by Fourier-transform infrared (FT-IR) spectrometry (Thermo Scientific Nicolet iS5). The binding energy was analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha). The amount of titanium ions immobilized on the surface of Ti⁴⁺-PA-cotton was analyzed by inductively coupled plasma-optical emission spectrophotometer (ICP-OES, Agilent Technologies 730 Series, USA). Thermogravimetric analyzer-differential scanning calorimeter (TG-DSC) data analyses were performed on a thermal analyzer (SDT-650, TA Instruments, USA) under air flow (mass flow, 100.00 mL/min) at a heating rate of 20°C/min from room temperature to 800°C.

2.4. Adsorptions of BSA, HRP, and β-Casein on Ti⁴⁺-PA-Cotton

The kinetic adsorption equilibrium experiment was performed to optimize the incubation time and study the adsorption mechanism. The initial adsorption rate and kinetic rate constant were determined based on the pseudo-second-order kinetic model (equation (1)), where Q_e (mg/g) and Q_t (mg/g) are the adsorption capacities at equilibrium and at time t (min), respectively; k₂ (g/mg·min) is the kinetic rate constant of the pseudo-second-order kinetic model. Besides, the adsorption isotherm experiment was conducted to identify the theoretical maximum adsorption capacity of Ti⁴⁺-PA-cotton for the target proteins based on the Langmuir model (equation (2)), where Q (mg/g) is the adsorption capacity after enrichment, Q_m (mg/g) is the saturated adsorption capacity (also known as theoretical maximum adsorption capacity), C_e (μg/mL) is the equilibrium concentration after enrichment, and K_L (L/mg) is the Langmuir constant.

2.5. Preparation of Ti⁴⁺-PA-Cotton SPE Pipette Tip

A piece of dry Ti⁴⁺-PA-cotton (50 mg) was fitted into the end of a pipette tip (1 mL, Axygen Scientific, Inc.) using a toothpick to prepare a lab-made SPE pipette tip (Figure S1). The Ti⁴⁺-PA-cotton SPE tip was washed with 1 mL of 0.1% TFA and conditioned with 1 mL of loading buffer twice before use.

2.6. Enrichment of Glycoproteins or Phosphoproteins in Standard Protein Mixtures

Ti⁴⁺-PA-cotton SPE pipette tip was applied for the enrichment of intact glycoproteins or phosphoproteins, respectively. The enrichment protocol was according to our previous report [31] and other reported work [24].

2.7. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein Mixtures

Simultaneous adsorption of both glycoproteins and phosphoproteins using Ti⁴⁺-PA-cotton SPE pipette tip was studied in standard protein mixtures, and the enrichment procedure is demonstrated in Figure 1.

Initially, 0.5 mL of protein mixtures (BSA, HRP, and β-casein with the same concentration of 1.0 mg/mL) were mixed with 0.5 mL of loading buffer (ACN/H₂O/TFA, 90: 8: 2, v/v/v). The mixture was pipetted up and down for 10 times in 2 min and incubated for 20 min at 4°C, and the fraction part was stored after incubation. The SPE pipette tip was washed three times with washing buffer (ACN/H₂O/TFA, 90: 9.9: 0.1, v/v/v) to remove the nonspecific proteins. Subsequently, the enriched glycoproteins and phosphoproteins were eluted step by step with different elution buffers. The enriched glycoproteins were eluted with 100 μL of elution buffer I (ACN/H₂O/TFA, 30: 69.9: 0.1, v/v, pH 2) for 2 h, and then the enriched phosphoproteins were eluted with 100 μL of elution buffer II (ammonium hydroxide, 10% v/v, pH 11.0) for 2 h. Both glycoproteins and phosphoproteins were eluted three times. Finally, the eluate I and eluate II were separately collected and concentrated for SDS-PAGE analysis. SDS-PAGE was operated in the Bio-Rad electrophoresis system (Bio-Rad Laboratories), and SDS-PAGE pattern was obtained by Amersham™ Imager 600 (GE Healthcare Life Sciences).

2.8. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Biological Samples

Ti⁴⁺-PA-cotton SPE pipette tip was further applied in the biological system of human serum. Human blood donated from the healthy donors was collected after the subsequent centrifugation at 10,000 rpm for 10 min, and then the serum was diluted to 50-fold using Tris-HCl buffer (pH 7.4) and spiked with HRP and β-casein, both at a concentration of 1 mg/mL. Then, 250 μL of spiked human serum was mixed with 250 μL of loading buffer and incubated with Ti⁴⁺-PA-cotton SPE tip for 20 min at 4°C. The supernatant, eluate I, and eluate II were analyzed by SDS-PAGE after enrichment procedure.

2.9. The Regeneration of Ti⁴⁺-PA-Cotton

The reuse of Ti⁴⁺-PA-cotton SPE pipette tip was also studied, the SPE tip which was used for six times enrichment was regenerated, and then it was applied for enrichment operations. The regeneration method was briefly described in the SM.

3. Results and Discussion

3.1. Characterization of Ti⁴⁺-PA-Cotton

The Ti⁴⁺-PA-cotton was specifically designed in this work for the enrichment of glycoproteins and phosphoproteins. On the one hand, as a macromolecular polysaccharide, cellulose is the main component of natural cotton, and cotton could be used directly as a HILIC adsorption material due to the functional hydroxyl of cellulose. On the other hand, abundant phosphate groups could be introduced by PA modification through one-step assembly reaction and further chelated with titanium cations to form an IMAC adsorption material. Thus, Ti⁴⁺-PA-cotton had the dual function of HILIC and IMAC materials.

The morphological structures of the unmodified degrease cotton, PA-cotton, and Ti⁴⁺-PA-cotton were studied by SEM. As shown in Figures 2(a) and 2(b), the degrease cotton had a relatively smooth surface, and the surface of PA-cotton (Figures S2A and S2B) and Ti⁴⁺-PA-cotton (Figures 2(c) and 2(d)) became rough after modification. This clearly showed that PA was successfully modified onto the surface of cotton. EDS elemental mapping results of the selected area displayed that the elements C, N, O, P, and Ti were distributed homogeneously on the surface of Ti⁴⁺-PA-cotton, indicating the successful immobilization of titanium cations (Figures 2(e)–2(j)). Elemental composition of Ti⁴⁺-PA-cotton was characterized by EDS spot analysis, the titanium ions immobilized on the surface of PA-cotton was 0.32% (wt%) and 0.09% (atom%), respectively (Figure S3 and Table S1).

Functional groups of degrease cotton and the prepared PA-Cotton were analyzed by FT-IR spectrometry (Figure S4). Compared with the FT-IR spectrum of cotton (Figure S4A), an asymmetric stretching vibration absorption band of O–P–O at 1633.41 cm⁻¹ could be observed in the spectrum of PA-Cotton (Figure S4B), the new peak ascribed to ν_as (O–P–O) indicated the successful modification of PA onto the surface of the degrease cotton [32].

To obtain the information of the chemical state and surface chemical composition of Ti⁴⁺-PA-cotton, XPS measurement was carried out. As shown in Figure 3, the survey spectrum indicated the peaks of C, N, O, P, and Ti element in Ti⁴⁺-PA-cotton, which were in good agreement with the elemental composition. The Ti 2p spectrum in the inset demonstrated that the binding energy of Ti 2p3/2 and Ti 2p1/2 was at peaks of 458.8 and 464.1 eV, indicating that titanium remained in the oxidation state of IV [33].

The element content of titanium in Ti⁴⁺-PA-cotton was determined by ICP-OES and calculated to be 5601.7 mg/kg, and the ICP-OES results also proved that there was titanium element in Ti⁴⁺-PA-cotton.

TG-DSC analyses were employed to estimate the weight ratio of the inorganic and organic components in Ti⁴⁺-PA-Cotton. Figure S5 shows that the weight loss curve of degreased cotton and Ti⁴⁺-PA-cotton was nearly same from room temperature to 100°C, which was due to the loss of water. TG curve of Ti⁴⁺-PA-cotton (red solid line) dropped sharply with the increasing temperature (from 100 to 365°C) comparing with that of degrease cotton (black solid line). The residual percent of degrease cotton at 800°C was 4.679%, while the value of Ti⁴⁺-PA-cotton was 5.074%. The percent difference was ascribed to the existence of titanium ions, demonstrating the successful functionalization of PA and titanium ions.

3.2. Adsorptions of BSA, HRP, and β-Casein on Ti⁴⁺-PA-Cotton

The adsorption kinetics were studied to determine the appropriate incubation time for extraction by Ti⁴⁺-PA-Cotton. As exhibited in Figure 4(a), the curve of BSA slightly declined due to the nonspecific adsorption in the initial stage; however, the curve was quite flat compared to that of HPR and β-casein. The supernatant concentration decreased as the incubation time increased in HRP and β-casein. As for HRP curve, the adsorption rate was fast within 20 min and then the curve came to a plateau. As for β-casein curve, the equilibrium time was 15 min, thus, 20 min would be an appropriate time for incubation. Besides, the kinetic rate constant was determined based on the pseudo-second-order kinetic model, which could reflect the adsorption speed of Ti⁴⁺-PA-cotton for the target proteins. As shown in Figure 4(b), the experimental data fitted well with the pseudo-second-order kinetic model with high regression coefficients. So, the adsorption process was mainly controlled by the chemical adsorption mechanism [34, 35]. The initial adsorption rate ν₀ was the reciprocal of the intercept of the fitted curve, which was 259.1 mg/g·min for β-casein, 63.1 mg/g·min for HRP and 45.8 mg/g·min for BSA, respectively. The adsorption rates of β-casein and HRP were higher than that of BSA, suggesting the selective adsorption property of Ti⁴⁺-PA-cotton towards targeted proteins. The kinetic rate constant k₂ and theoretical equilibrium capacity were calculated based on the slope and intercept of the fitted curve and listed in Table S2.

(a)

(b)

(c)

(d)

Adsorption isotherm experiments were carried out to determine the adsorption capacity and binding ability. As shown in Figure 4(c), as the initial concentration increased, the adsorption capacities increased at first and reached a plateau at last. The adsorption isotherms of β-casein were highest, and the adsorption isotherms of BSA were lowest. The adsorption capacity of the targeted proteins (β-casein and HRP) was much higher than that of BSA at the same concentration. The enrichment ratio (ER%) was determined by the mass ratio of the adsorbed targeted proteins to the total proteins. As shown in Figure S6, the ER% was more than 97% when the concentration was within 800 μg/mL. However, the ER% declined when the concentration was beyond 800 μg/mL, and this phenomenon was because that the added Ti⁴⁺-PA-cotton were not sufficient to adsorb β-casein completely, which could also be observed in the plot of HRP enrichment ratio.

The adsorption behavior was fitted with the Langmuir model with the regression coefficients (R²) higher than 0.99 (Figure 4(d)), indicating that the adsorption was monolayer adsorption [36]. The maximum adsorption capacities of Ti⁴⁺-PA-cotton for β-casein, HRP, and BSA were calculated to be 833.3 mg/g, 384.6 mg/g, and 82.3 mg/g, respectively. The results confirmed that Ti⁴⁺-PA-cotton possessed excellent enrichment ability for phosphoproteins and glycoproteins. Comparing the two targeted proteins, the adsorption rate and maximum capacity of Ti⁴⁺-PA-cotton for β-casein (259.1 mg/g·min, 833.3 mg/g) was higher than those of HPR (63.1 mg/g·min, 384.6 mg/g). The result was in accordance with their own enrichment mechanism, i.e., the coordination binding of phosphoprotein enrichment was stronger than HILIC binding of glycoprotein enrichment.

3.3. Enrichment of Glycoproteins or Phosphoproteins in Standard Protein Mixtures

The feasibility of phosphoprotein enrichment using Ti⁴⁺-PA-cotton SPE pipette tip was validated in the standard protein mixtures of β-casein and BSA with the mass ratio of 1 : 1. As shown in the SDS-PAGE pattern of Figure 5, the lane of IPM1, S1, and E1 represented the initial protein mixture, supernatant, and eluate after phosphoprotein enrichment. In the lane of IPM1, the bands of BSA and β-casein could be clearly observed. In the S1 lane, only the band of BSA appeared, while the band of β-casein disappeared. In lane E1, the band of β-casein reappeared and could be obviously observed, which showed that phosphoprotein β-casein was adsorbed by Ti⁴⁺-PA-cotton and desorbed into the elution. The experimental results proved that Ti⁴⁺-PA-cotton SPE tip had a satisfactory phosphoprotein enrichment ability.

The feasibility of glycoprotein enrichment using Ti⁴⁺-PA-cotton SPE pipette tip was also studied in the standard protein mixture of HRP and BSA with the mass ratio of 1 : 1. The lane of IPM2, S2, and E2 represented the initial protein mixture, supernatant, and eluate after glycoprotein enrichment. There were two obvious bands of BSA and HRP in IPM2 lane, and the band of HRP almost faded in the lane of S2 and reappeared in lane of E2 (Figure 5). The SDS-PAGE pattern verified that Ti⁴⁺-PA-cotton SPE pipette tip could enrich the glycoproteins as well.

3.4. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein Mixtures

In consideration of the excellent enrichment capability of Ti⁴⁺-PA-cotton SPE pipette tip for phosphoproteins together with glycoproteins, the prepared SPE tip was also applied for the selective and specific glycoprotein enrichment and phosphoprotein enrichment simultaneously. As shown in Figure 6, three bands in the lane of IPM represented the proteins of BSA, HRP, and β-casein, respectively. There were a light band of HRP and a dense band of BSA in lane S. The first eluate merely contained protein HRP with the molecular weight of 44 kDa in lane E1, and only β-casein band was present in lane E2 without other protein bands. The SDS-PAGE analyses indicated that Ti⁴⁺-PA-cotton SPE pipette tip had excellent enrichment specificity. Besides, the SPE tip could adsorb glycoproteins and phosphoproteins in a single step and release glycoproteins and phosphoproteins step by step using different elution buffers. The application of the Ti⁴⁺-PA-cotton SPE pipette tip could simplify the operation process, reduce enrichment time, and increase the enrichment efficiency. It is also important to mention that the SPE tip would play an important role in such a situation where the clinical samples were in low content and precious.

(a)

(b)

3.5. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Biological Samples

Human serum was frequently used for disease diagnosis, treatment or prognosis monitoring in clinical studies. Ti⁴⁺-PA-cotton SPE pipette tip was further applied in 50-fold diluted human serum spiked with 1 μg/μL HRP and 1 μg/μL β-casein. As we can see in Figure 6(b), the bands of HRP and β-casein in lane S were obscure compared to that in IPM lane. In lane E1, the band of HRP could be seen, demonstrating the successful enrichment and release of glycoprotein, as for the endogenous glycoproteins (transferrin, Trf and immunoglobulin G, IgG), the adsorption of HRP was stronger due to its relatively low steric hindrance. In addition, we observed only the band of β-casein in lane E2, which indicated that phosphoproteins were adsorbed onto the SPE tip during incubation process and desorbed during the second elution process. Besides, the highly abundant interfering protein HSA was eliminated in both E1 and E2. The results confirmed the excellent performance of Ti⁴⁺-PA-cotton SPE tip for the simultaneous adsorption of glycoproteins and phosphoproteins in biological samples.

3.6. The Regeneration of Ti⁴⁺-PA-Cotton

To evaluate the enrichment performance of the Ti⁴⁺-PA-cotton which was regenerated after six times utilization, standard protein mixture of BSA, HRP, and β-casein was used. As demonstrated in Figure S7, HRP and β-casein could be clearly observed in lane E1 and lane E2, which was similar to the newly prepared materials in Figure 6(a). This could be ascribed to the reloading of titanium ions onto the surface of PA-cotton. There was a little denser band of HRP in lane S compared to Figure 6(a), which indicated that the glycoprotein enrichment ability was slightly affected after reutilization. The adsorption ratio of the regenerated materials to the newly synthesized ones remained 90.0% for β-casein and 78.9% for HRP, respectively (Figure S8). This can be explained by the fact that the regeneration procedure only involved the reimmobilization of titanium ions but not the hydroxylation procedure of cotton. Nevertheless, the SPE tip after regeneration could still meet the requirements of glycoprotein and phosphoprotein enrichment simultaneously.

4. Conclusion

In this work, a facile approach for the preparation of dual-functional Ti⁴⁺-PA-Cotton was developed, and the Ti⁴⁺-PA-cotton SPE pipette tip was lab-made for the simultaneous adsorption of glycoproteins and phosphoproteins, which was validated by the analysis of SDS-PAGE. The maximum adsorption capacities of Ti⁴⁺-PA-cotton for β-casein and HRP were calculated to be 833.3 mg/g and 384.6 mg/g with the adsorption rate of 259.1 and 63.1 mg/g·min, respectively. The preparation method of Ti⁴⁺-PA-cotton was facile, low cost, and environmentally friendly; the SPE tip was easy to operate and beneficial for production expansion with great selectivity and sensitivity. This integrated approach will provide a bright and promising prospect for the enrichment and comprehensive analysis of different post-translational modified proteins.

Abbreviations

BSA:	Bovine serum albumin
HILIC:	Hydrophilic interaction liquid chromatography
HRP:	Horseradish peroxidase
IMAC:	Immobilized metal ion affinity chromatography
MOAC:	Metal oxide affinity chromatography
PA:	Phytic acid
PTM:	Post-translational modifications
SDS-PAGE:	Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SPE:	Solid phase extraction.

Data Availability

The data used to support the findings of this study are included in Supplementary Material.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Dawei Zhang and Zhenhua Zhou contributed equally to this work.

Acknowledgments

This work was financially supported by Key Research Center Construction Project of Shanghai Municipal Health Commission (2022ZZ01013) and Shanghai Science and Technology Commission Orthopedics Clinical Medical Research Center Construction Project (21MC1930100).

Supplementary Materials

The detailed experimental protocols, supplementary figures and tables, equations mentioned in the manuscript, and related references are included in Supplementary Materials. (Supplementary Materials)

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Copyright © 2023 Dawei Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Chemistry

Simultaneous Adsorption of Intact Glycoproteins and Phosphoproteins Using Hydrophilic Titanium (IV) Ion-Immobilized Cotton Fiber Functionalized by Phytic Acid Assembly

Abstract

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Preparation of Ti4+-PA-Cotton

2.3. Characterization of Ti4+-PA-Cotton

2.4. Adsorptions of BSA, HRP, and β-Casein on Ti4+-PA-Cotton

2.5. Preparation of Ti4+-PA-Cotton SPE Pipette Tip

2.6. Enrichment of Glycoproteins or Phosphoproteins in Standard Protein Mixtures

2.7. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein Mixtures

2.8. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Biological Samples

2.9. The Regeneration of Ti4+-PA-Cotton

3. Results and Discussion

3.1. Characterization of Ti4+-PA-Cotton

3.2. Adsorptions of BSA, HRP, and β-Casein on Ti4+-PA-Cotton

3.3. Enrichment of Glycoproteins or Phosphoproteins in Standard Protein Mixtures

3.4. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein Mixtures

3.5. Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Biological Samples

3.6. The Regeneration of Ti4+-PA-Cotton

4. Conclusion

Abbreviations

Data Availability

Conflicts of Interest

Authors’ Contributions

Acknowledgments

Supplementary Materials

References

Copyright

2.2. Preparation of Ti⁴⁺-PA-Cotton

2.3. Characterization of Ti⁴⁺-PA-Cotton

2.4. Adsorptions of BSA, HRP, and β-Casein on Ti⁴⁺-PA-Cotton

2.5. Preparation of Ti⁴⁺-PA-Cotton SPE Pipette Tip

2.9. The Regeneration of Ti⁴⁺-PA-Cotton

3.1. Characterization of Ti⁴⁺-PA-Cotton

3.2. Adsorptions of BSA, HRP, and β-Casein on Ti⁴⁺-PA-Cotton

3.6. The Regeneration of Ti⁴⁺-PA-Cotton