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

Protein glycosylation and phosphorylation are two important post-translational modifcations and play signifcant roles in various biological processes. However, both glycoproteins and phosphoproteins are in low abundance in complex samples


Introduction
Glycosylation and phosphorylation, two ubiquitous posttranslational modifcations (PTMs) of proteins, play signifcant 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][2][3]. Protein glycosylation and phosphorylation were involved in the numerous biological events, such as gene expression, molecular recognition, signaling transduction, and immune response [4][5][6]. Aberrant glycosylation has been proven to be related to many human diseases, such as cardiovascular disease, cancer, and congenital disorders [7,8]. Te majority of the currently available FDA-approved tumor biomarkers are glycoproteins. Meanwhile, phosphoprotein dysfunction was also verifed 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. Tus, 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 specifc enrichment of glycoproteins and phosphoproteins were mutually independent. For glycoprotein/glycopeptide enrichment, lectin-afnity chromatography [12], boronic acid afnity 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 afnity chromatography (IMAC) and metal oxide afnity chromatography (MOAC) were the two main enrichment strategies due to their merits of low cost and ease-of-use [16][17][18][19].
Although great achievements have been made in the feld 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 sufer from problems when the amount of biological sample was low and precious. Tus, a strategy for the simultaneous enrichment of phosphorylated and glycosylated proteins was proposed. Te 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 diferent enrichment conditions when enriching diferent 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][25][26][27][28], and these research studies focused on the enrichment of the peptides rather than the intact proteins. Te 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 diferent PTMs. Terefore, more research eforts are still needed to develop new materials and integrated approaches for their simultaneous enrichment with high selectivity and specifcity by a facile approach.
In this work, we prepared a novel composite material consisting of a cotton fber functionalized with phytic acid and immobilized titanium (IV) ions (denoted as Ti 4+ -PAcotton) for the simultaneous enrichment of glycoproteins and phosphoproteins in a single step, and then the enriched glycoproteins and phosphoproteins could be eluted sequentially using diferent elution bufers. To the best of our knowledge, there are few studies aiming at the simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. Te Ti 4+ -PA-cotton with unique architecture was specifcally 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. Tus, 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 modifed onto the surface of diferent materials through one-step assembly reaction. Te assembly was triggered via various forces mainly dominated by hydrogen bond [30]. Te six phosphate groups of PA molecule could provide abundant afnity sites for the immobilization of titanium ions, which were able to coordinate with the phosphate groups of phosphoproteins. Te prepared Ti 4+ -PA-cotton was nontoxic, biodegradable, biocompatible, and environmentally friendly. In addition, Ti 4+ -PA-cotton was ftted into a pipette tip for the solid phase extraction (SPE) of glycoproteins and phosphoproteins. Te SPE approach based on Ti 4+ -PA-cotton combined HILIC strategy and IMAC strategy, which could reduce enrichment time, facilitate enrichment operation, and was favorable for its application and promotion. Te adsorptions of BSA, HRP, and β-casein on Ti 4+ -PA-cotton were studied. Te enrichment performance of Ti 4+ -PA-cotton SPE tip was validated by SDS-PAGE, which demonstrated a promising prospect in scientifc research and clinical study of glycoproteins and phosphoproteins. . 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 bufer 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 purifcation.

Preparation of Ti 4+ -PA-Cotton.
Te facile synthetic procedure of Ti 4+ -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 Ti 4+ -PA-cotton. Detailed preparation protocols were described in the supplementary material (SM).

Characterization of Ti 4+ -PA-Cotton.
Ti 4+ -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 Ti 4+ -PA-cotton was studied by SEM equipped with energy-dispersive spectrometry (EDS) microanalysis unit. Te functional groups of Ti 4+ -PA-cotton were analyzed by Fourier-transform infrared (FT-IR) spectrometry (Termo Scientifc Nicolet iS5). Te binding energy was analyzed by X-ray photoelectron spectroscopy (XPS, Termo Scientifc K-Alpha). Te amount of titanium ions immobilized on the surface of Ti 4+ -PA-cotton was analyzed by inductively coupled plasmaoptical emission spectrophotometer (ICP-OES, Agilent Technologies 730 Series, USA). Termogravimetric analyzer-diferential scanning calorimeter (TG-DSC) data analyses were performed on a thermal analyzer (SDT-650, TA Instruments, USA) under air fow (mass fow, 100.00 mL/min) at a heating rate of 20°C/min from room temperature to 800°C.

Adsorptions of BSA, HRP, and β-Casein on Ti 4+ -PA-
Cotton. Te kinetic adsorption equilibrium experiment was performed to optimize the incubation time and study the adsorption mechanism. Te initial adsorption rate and kinetic rate constant were determined based on the pseudosecond-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; k 2 (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 Ti 4+ -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.

Preparation of Ti 4+ -PA-Cotton SPE Pipette Tip.
A piece of dry Ti 4+ -PA-cotton (50 mg) was ftted into the end of a pipette tip (1 mL, Axygen Scientifc, Inc.) using a toothpick to prepare a lab-made SPE pipette tip ( Figure S1). Te Ti 4+ -PA-cotton SPE tip was washed with 1 mL of 0.1% TFA and conditioned with 1 mL of loading bufer twice before use.

Enrichment of Glycoproteins or Phosphoproteins in Standard Protein
Mixtures. Ti 4+ -PA-cotton SPE pipette tip was applied for the enrichment of intact glycoproteins or phosphoproteins, respectively. Te enrichment protocol was according to our previous report [31] and other reported work [24].

Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein
Mixtures. Simultaneous adsorption of both glycoproteins and phosphoproteins using Ti 4+ -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 bufer (ACN/H 2 O/TFA, 90: 8: 2, v/v/v). Te 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. Te SPE pipette tip was washed three times with washing bufer (ACN/H 2 O/TFA, 90: 9.9: 0.1, v/v/v) to remove the nonspecifc proteins. Subsequently, the enriched glycoproteins and phosphoproteins were eluted step by step with diferent elution bufers. Te enriched glycoproteins were eluted with 100 μL of elution bufer I (ACN/H 2 O/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 bufer 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).

Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Biological
Samples. Ti 4+ -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 50fold using Tris-HCl bufer (pH 7.4) and spiked with HRP and β-casein, both at a concentration of 1 mg/mL. Ten, 250 μL of spiked human serum was mixed with 250 μL of loading bufer and incubated with Ti 4+ -PA-cotton SPE tip for 20 min at 4°C. Te supernatant, eluate I, and eluate II were analyzed by SDS-PAGE after enrichment procedure.

Te
Regeneration of Ti 4+ -PA-Cotton. Te reuse of Ti 4+ -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. Te regeneration method was briefy described in the SM.

Characterization of Ti 4+ -PA-Cotton.
Te Ti 4+ -PA-cotton was specifcally 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 modifcation through one-step assembly reaction and further chelated with titanium cations to form an IMAC adsorption material. Tus, Ti 4+ -PA-cotton had the dual function of HILIC and IMAC materials.
Te morphological structures of the unmodifed degrease cotton, PA-cotton, and Ti 4+ -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 Ti 4+ -PA-cotton (Figures 2(c) and 2(d)) became rough after modifcation. Tis clearly showed that PA was successfully modifed 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 Ti 4+ -PAcotton, indicating the successful immobilization of titanium cations (Figures 2(e)-2(j)). Elemental composition of Ti 4+ -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 ] as (O-P-O) indicated the successful modifcation of PA onto the surface of the degrease cotton [32].
To obtain the information of the chemical state and surface chemical composition of Ti 4+ -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 Ti 4+ -PA-cotton, which were in good agreement with the elemental composition. Te 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].
Te element content of titanium in Ti 4+ -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 Ti 4+ -PA-cotton.
TG-DSC analyses were employed to estimate the weight ratio of the inorganic and organic components in Ti 4+ -PA-Cotton. Figure S5 shows that the weight loss curve of degreased cotton and Ti 4+ -PA-cotton was nearly same from room temperature to 100°C, which was due to the loss of water. TG curve of Ti 4+ -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). Te residual percent of degrease cotton at 800°C was 4.679%, while the value of Ti 4+ -PA-cotton was 5.074%. Te percent diference was ascribed to the existence of titanium ions, demonstrating the successful functionalization of PA and titanium ions.

Adsorptions of BSA, HRP, and β-Casein on Ti 4+ -PA-Cotton.
Te adsorption kinetics were studied to determine the appropriate incubation time for extraction by Ti 4+ -PA-Cotton. As exhibited in Figure 4(a), the curve of BSA slightly declined due to the nonspecifc adsorption in the initial stage; however, the curve was quite fat compared to that of HPR and β-casein. Te 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 refect the adsorption speed of Ti 4+ -PA-cotton for the target proteins. As shown in Figure 4(b), the experimental data ftted well with the pseudo-second-order kinetic model with high regression coefcients. So, the adsorption process was mainly controlled by the chemical adsorption mechanism [34,35]. Te initial adsorption rate ] 0 was the reciprocal of the intercept of the ftted 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. Te adsorption rates of β-casein and HRP were higher than that of BSA, suggesting the selective adsorption property of Ti 4+ -PA-cotton towards targeted proteins. Te kinetic rate constant k 2 and theoretical equilibrium capacity were calculated based on the slope and intercept of the ftted curve and listed in Table S2.
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 frst and reached a plateau at last. Te adsorption isotherms of β-casein were highest, and the adsorption isotherms of BSA were lowest. Te adsorption capacity of the targeted proteins (β-casein and HRP) was much higher than that of BSA at the same concentration. Te 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 Ti 4+ -PA-cotton were not sufcient to adsorb β-casein completely, which could also be observed in the plot of HRP enrichment ratio.
Te adsorption behavior was ftted with the Langmuir model with the regression coefcients (R 2 ) higher than 0.99 (Figure 4(d)), indicating that the adsorption was monolayer adsorption [36]. Te maximum adsorption capacities of Ti 4+ -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. Te results confrmed that Ti 4+ -PA-cotton possessed excellent enrichment ability for phosphoproteins and glycoproteins. Comparing the two targeted proteins, the adsorption rate and maximum capacity of Ti 4+ -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). Te 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.

Enrichment of Glycoproteins or Phosphoproteins in Standard Protein
Mixtures. Te feasibility of phosphoprotein enrichment using Ti 4+ -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 Ti 4+ -PA-cotton and desorbed into the elution. Te experimental results proved that Ti 4+ -PA-cotton SPE tip had a satisfactory phosphoprotein enrichment ability.
Te feasibility of glycoprotein enrichment using Ti 4+ -PA-cotton SPE pipette tip was also studied in the standard protein mixture of HRP and BSA with the mass ratio of 1 : 1. Te lane of IPM2, S2, and E2 represented the initial protein mixture, supernatant, and eluate after glycoprotein enrichment. Tere 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). Te SDS-PAGE pattern verifed that Ti 4+ -PA-cotton SPE pipette tip could enrich the glycoproteins as well.

Simultaneous Adsorption of Glycoproteins and Phosphoproteins in Standard Protein
Mixtures. In consideration of the excellent enrichment capability of Ti 4+ -PA-cotton SPE pipette tip for phosphoproteins together with glycoproteins, the prepared SPE tip was also applied for the selective and specifc 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. Tere were a light band of HRP and a dense band of BSA in lane S. Te frst 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. Te SDS-PAGE analyses indicated that Ti 4+ -PA-cotton SPE pipette tip had excellent enrichment specifcity. Besides, the SPE tip could adsorb glycoproteins and phosphoproteins in a single step and release glycoproteins and phosphoproteins step by step using diferent elution bufers. Te application of the Ti 4+ -PA-cotton SPE pipette tip could simplify the operation process, reduce enrichment time, and increase the enrichment efciency. 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.  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. Te results confrmed the excellent performance of Ti 4+ -PA-cotton SPE tip for the simultaneous adsorption of glycoproteins and phosphoproteins in biological samples.

Te Regeneration of Ti 4+ -PA-Cotton.
To evaluate the enrichment performance of the Ti 4+ -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). Tis could be   ascribed to the reloading of titanium ions onto the surface of PA-cotton. Tere was a little denser band of HRP in lane S compared to Figure 6(a), which indicated that the glycoprotein enrichment ability was slightly afected after reutilization. Te 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). Tis 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.

Conclusion
In this work, a facile approach for the preparation of dual-functional Ti 4+ -PA-Cotton was developed, and the Ti 4+ -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. Te maximum adsorption capacities of Ti 4+ -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. Te preparation method of Ti 4+ -PAcotton was facile, low cost, and environmentally friendly; the SPE tip was easy to operate and benefcial for