Hydrophilic Phytic Acid-Coated Magnetic Graphene for Titanium(IV

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Hydrophilic Phytic Acid-Coated Magnetic Graphene for Titanium(IV) Immobilization as a Novel HILIC-IMAC Platform for Glycopeptides and Phosphopeptides Enrichment with Controllable Selectivity Yayun Hong, Hongli Zhao, Chenlu Pu, Qiliang Zhan, Qianying Sheng, and Minbo Lan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02614 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Analytical Chemistry

Hydrophilic Phytic Acid-Coated Magnetic Graphene for Titanium(IV) Immobilization as a Novel HILIC-IMAC Platform for Glycopeptides and Phosphopeptides Enrichment with Controllable Selectivity Yayun Hong,† Hongli Zhao,*† Chenlu Pu,† Qiliang Zhan,† Qianying Sheng† and Minbo Lan*†,‡ †

Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China ‡

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China. Corresponding authors’ E-mail: [email protected]; [email protected].

ABSTRACT: In this work, multifunctional Ti4+-immobilized phytic acid modified magnetic graphene (denoted as MagG@PEI@PA-Ti4+) nanocomposites were fabricated through a facile route for simultaneous/respective enrichment of Nglycopeptides and phosphopeptides. Phytic acid (PA), with six phosphate groups, possesses excellent hydrophilicity and metal ion coordination ability, which endowed the MagG@PEI@PA-Ti4+ with combined properties of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based materials. Based on the different binding ability of N-glycopeptides and phosphopeptides on MagG@PEI@PA-Ti4+, the MagG@PEI@PA-Ti4+ nanocomposites could enrich N-glycopeptides and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glycopeptides and phosphopeptides. And the proposed nanocomposites demonstrated an outstanding performance for selective enrichment of N-glycopeptides (selectivity: 1:1000 molar ratios of IgG/BSA; sensitivity: 0.5 fmol/µL IgG; loading capacity: 300 mg g-1; recovery: >90%) and phosphopeptides (selectivity: 1:5000 molar ratios of α-casein/BSA; sensitivity: 0.1 fmol/µL α-casein; loading capacity: 100 mg g-1; recovery: >90%). Taking advantage of these merits, a total of 393 Nglycopeptides derived from 259 glycoproteins and 574 phosphopeptides derived from 341 phosphoproteins were identified from 200 µg of HeLa cell extracts through a single-step enrichment using MagG@PEI@PA-Ti4+.

Protein glycosylation and phosphorylation, two of the most common post-translational modifications (PTMs), are involved in many important biological processes, such as metabolism, signal transduction and immune defense.1-3 Abnormal glycosylation and phosphorylation are closely associated with many diseases,4-7 which may contain potential biomarkers with clinical significance. Simultaneous monitoring and accurate analysis of glycosylation and phosphorylation are of great significance for the discovery of disease biomarker. Annoyingly, although mass spectrometry (MS) based techniques are the most powerful platform for protein PTMs research,8,9 glycopeptides/phosphopeptides in biological samples still cannot be identified directly by MS analysis due to their low abundance and the extremely complex components of biological samples. Therefore, an effective enrichment prior to MS analysis is a prerequisite for accurate identification of glycopeptides/phosphopeptides. Currently, various strategies have been developed for glycopeptides or phosphopeptides enrichment, such as lectin affinity chromatography,10,11 covalent capture technology 12-14 and hydrophilic interaction chromatography (HILIC) 15-17 for glycopeptides, while immobilized metal affinity chromatography (IMAC) 18-20 and metal oxide affinity chromatography (MOAC) 21-23 for phosphopeptides. However, these methods or materials can only be used to enrich glycopeptides or phosphopeptides individually. In attempts to identify glycopeptides

and phosphopeptides simultaneously in a sample, it is necessary to respectively enrich N-glycopeptides and phosphopeptides by using different materials in parallel.24,25 At present, few materials can enrich glycopeptides and phosphopeptides simultaneously or respectively through a facile operation. In addition, the tedious manipulations and harsh conditions in the synthesis of many materials not only make the synthetic process time-consuming, but also result in a low functionalization efficiency. Therefore, it is an inevitable trend to develop a facile method to synthesize novel materials for simultaneous/respective enrichment of glycopeptides and phosphopeptides from one sample. HILIC, which is based on the hydrophilic interaction between glycopeptides and hydrophilic materials, has been regarded as the most popular method for glycopeptides enrichment. HILIC materials with excellent hydrophilicity can effectively reduce the non-specific adsorption. Polyethyleneimine (PEI), a kind of water-soluble polymer, can be used as a perfect candidate to construct HILIC materials due to their high density of amino groups for easy post-functionalization and excellent hydrophilicity.26,27 IMAC, which relies on the reversible affinity of the phosphate groups to immobilized metal ions, is one of the most widely used techniques for phosphopeptides enrichment. However, traditional chelating ligands, iminodicitic acid (IDA) and nitrilotriacetic acid (NTA), always result in the undesirable loss of the immobilized metal ions

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during sample loading and washing due to the relatively weaker interaction (each metal ion coordinates with only one IDA or NTA ligand).28-30 Thus, chelating ligands with higher chelating ability are desired. Phytic acid (PA), a natural and eco-friendly compound, possesses excellent hydrophilicity and metal ion coordination ability.31-33 The PA molecule, composed of six phosphate groups, could provide numerous affinity sites for metal ions immobilization. Besides, its excellent hydrophilicity can further reduce the non-specific adsorption. Therefore, PA with the unique structure and excellent hydrophilicity is considered as an ideal chelating ligand to prepare IMAC materials. Herein, Ti4+-immobilized phytic acid modified magnetic graphene (denoted as MagG@PEI@PA-Ti4+) nanocomposites, which possess combined properties of IMAC- and HILICbased materials, were synthesized through a facile route. Firstly, PEI modified magnetic graphene (MagG@PEI) nanocomposites were synthesized via a one-pot solvothermal method. The MagG@PEI nanocomposites possessed combined properties of a large surface area of graphene, magnetic property of Fe3O4 and excellent hydrophilicity of PEI. Due to the high density of amino groups of PEI, MagG@PEI exhibited a positive surface charge. Therefore, PA with negative charges could be easily grafted onto the surface of MagG@PEI through electrostatic interaction, followed by immobilizing Ti4+ on PA to form MagG@PEI@PA-Ti4+. Due to the large amount of Ti4+ and excellent hydrophilicity of PEI and PA, MagG@PEI@PA-Ti4+ could enrich N-glycopeptides and phosphopeptides simultaneously or respectively by using different enrichment conditions according to the different binding ability of N-glycopeptides and phosphopeptides. To the best of our knowledge, this is the first time PA has been used to modify material for effective enrichment of N-glycopeptides and phosphopeptides simultaneously by chelating with Ti4+. Furthermore, MagG@PEI@PA-Ti4+ showed a great advantage in identifying a lot of low abundance N-glycopeptides and phosphopeptides from a small amount of HeLa cell extracts. EXPERIMENTAL SECTION

Materials and Chemicals. Ferric chloride (FeCl3), sodium citrate, sodium acetate (NaAc), ethylene glycol, polyethyleneimine (PEI, 50%, Mw=70000), phytic acid (PA, 70 %) and Ti(SO4)2 were purchased from Aladdin (Shanghai, China). Ammonium bicarbonate (NH4HCO3), urea, α-casein (from bovine milk), bovine serum albumin (BSA), immunoglobulin G from human serum (IgG), dithiothreitol (DTT), iodoacetamide (IAA), trypsin and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNGase F was purchased from New England Biolabs (Ipswich, MA, USA). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were chromatographic grade. Water used in experiments was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents used in this research were at least of analytical grade without further purification. Synthesis of MagG@PEI@PA-Ti4+ Nanocomposites. MagG@PEI were synthesized via a solvothermal method. Briefly, 0.1 g FeCl3, 0.04 g sodium citrate and 0.5 g PEI were first dissolved in 65 mL ethylene glycol. Then, 0.1 g graphene was added and ultrasonicated for 1 h. Subsequently, 0.4 g NaAc was added with stirring. The mixture was stirred vigorously for 30 min and then transferred into a Teflon-lined stainless-steel autoclave (100 mL capacity). The autoclave was heated at 200 °C for 10 h. The obtained MagG@PEI nano-

composites were washed with deionized water and ethanol for several times, and then lyophilized to dryness. PA and Ti4+ were deposited onto the surface of the MagG@PEI nanocomposites via electrostatic interactions. The dried MagG@PEI was dispersed in 100 mL PA solution (7 mg mL-1) by 5 min ultrasonication. Then the mixture was mechanically stirred for 6 h at room temperature. The products (MagG@PEI@PA) were collected using a magnet and washed with deionized water for three times. Finally, the MagG@PEI@PA was incubated in 50 mL Ti(SO4)2 solution (100 mM) for 2 h to immobilize Ti4+. The obtained products (MagG@PEI@PA-Ti4+) were washed with deioned water for several times and then lyophilized to dryness. Characterization. Transmission electron microscopy (TEM) images were recorded on a JEOL JEM-1400 transmission electron microscope (JEOL, Japan). Scanning electron microscope (SEM) images were obtained using an S-3400N scanning electron microscope (Hitachi, Japan). The elemental mapping patterns were carried out using a XM-2 EDS (EDAX, USA) equipped to a Tecnai G2 F30 S-TWIN field emission transmission electron microscope (FEI, USA). Energy dispersive spectroscopy (EDS) images were obtained by Falion 60S (EDAX, USA). Fourier transform infrared (FT-IR) spectra were performed on Nicolet 6700 (Thermo Fisher Scientific, USA) using KBr pellets. Zeta potential measurements were performed on a Nano-ZS90 instrument (Malvern, UK) in water at 25 oC. The magnetic hysteresis curves were measured by Lakeshore 7407 vibrating sample magnetometer (Lakeshore, USA) at room temperature. Sample Preparation. 1 mg α-casein was dissolved in 1 mL NH4HCO3 buffer (50 mM, pH 8.2) and digested at 37 oC for 17 h with trypsin at an enzyme to protein ratio of 1:25 (w/w). 1 mg IgG (or BSA) was dissolved in 100 µL NH4HCO3 buffer (50 mM, pH 8.2) containing 8 M urea. Subsequently, 5 µL DTT (200 mM) was add to reduce the denatured protein samples at 56 oC for 45 min. Then 20 µL IAA (200 mM) was added and the mixtures were incubated in dark for 30 min. Finally, the mixture was diluted to 1 mL with NH4HCO3 buffer and treated with trypsin at 37 oC for 17 h at an enzyme to protein ratio of 1:25 (w/w). HeLa cells were washed twice with ice-cold PBS buffer (pH 7.4) and then lysed with lysis buffer (50 mM Tris-HCl, 8 M urea, 2% protease cocktail, 1% Triton X-100, 1 mM C3H7Na2PO6, 1 mM Na4P2O7, 1 mM NaF, and 1 mM Na3VO4, pH 7.4). The supernatant was collected by centrifugation. Afterwards, five volume of precipitation solution (acetone/ethanol/acetic acid = 50/50/50, v/v/v) was added to precipitate the protein. The protein precipitate was collected by centrifugation and resuspended in 50 mM NH4HCO3 buffer. The protein concentration was measured by Bradford assay. The process of protein digestion was the same as that of IgG. Finally, the tryptic digest was lyophilized for further use. N-Glycopeptides and Phosphopeptides Enrichment. For glycopeptides enrichment, 20 µg of MagG@PEI@PA-Ti4+ nanocomposites were dispersed in 200 µL of loading buffer (ACN/H2O/TFA =90/8/2, v/v/v) containing a certain amount of IgG digest and then incubated at room temperature for 30 min. After that, the nanocomposites were collected with a magnet and rinsed with 100 µL of washing buffer (ACN/H2O/TFA =90/9.9/0.1, v/v/v) for three times. Subsequently, the captured glycopeptides were eluted with 10 µL 3% TFA aqueous solution for 15 min. The eluent was analyzed by MALDI-TOF MS.


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Analytical Chemistry For phosphopeptides enrichment, 20 µg of MagG@PEI@PA-Ti4+ nanocomposites were dispersed in 200 µL of loading buffer (ACN/H2O/TFA =50/48/2, v/v/v) containing a certain amount of α-casein digest and then incubated at room temperature for 30 min. After that, the nanocomposites were collected with a magnet and rinsed with 100 µL of washing buffer (ACN/H2O/TFA =50/49.9/0.1, v/v/v) for three times. Subsequently, the captured phosphopeptides were eluted with 10 µL NH4OH solution (0.4 M) for 15 min. The eluent was analyzed by MALDI-TOF MS. For glycopeptides and phosphopeptides mixture enrichment, the mixture of IgG digest and α-casein digest with a molar ratio of 1:1 was diluted with 200 µL of loading buffer (ACN/H2O/TFA =90/8/2, v/v/v). Then, 40 µg of MagG@PEI@PA-Ti4+ nanocomposites was added. After incubation at room temperature for 30 min, the nanocomposites were collected with a magnet and rinsed with 200 µL of washing buffer (ACN/H2O/TFA =90/9.9/0.1, v/v/v) for three times. Subsequently, the captured peptides were eluted with different elution buffer (10 µL for each elution buffer) and analyzed by MALDI-TOF MS. For HeLa cell extracts enrichment, 200 µg of tryptic digests were diluted with 500 µL of loading buffer (ACN/H2O/TFA =90/8/2, v/v/v) and incubated with 2 mg of MagG@PEI@PATi4+ nanocomposites for 30 min. Subsequently, the nanocomposites were collected and rinsed with 500 µL of washing buffer (ACN/H2O/TFA =90/9.9/0.1, v/v/v) for three times. The captured glycopeptides were eluted twice with 100 µL of elution buffer 1 (3% TFA aqueous solution), and then the captured phosphopeptides were eluted twice with 100 µL of elution buffer 2 (0.4 M NH4OH solution). The glycopeptides eluent was lyophilized and then deglycosylated for nano-LCMS/MS analysis. The phosphopeptides eluent was lyophilized for nano-LC-MS/MS analysis directly. Deglycosylation of N-Glycopeptides by PNGase F. The lyophilized glycopeptides were redissolved in 50 mM NH4HCO3, and 500 unites of PNGaseF was added. The mixture was incubated at 37 °C for 16 h. Finally, the solution was lyophilized for nano-LC-MS/MS analysis. Mass Spectrometry Analysis. MALDI-TOF MS experiments were performed on a 4800 Plus MALDI-TOF MS (AB Sciex, USA) in reflect positive ion mode. 1 µL of analyte was mixed with 1 µL of matrix (DHB, 25 mg/mL, ACN/H2O/H3PO4=70:29:1, v/v/v) and dropped on the plate for MALDI-TOF MS analysis. Nano-LC-MS/MS experiments were performed on an EASY-nLC 1000 Nano HPLC System (Thermo Fisher Scientific, Bremen, Germany) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Bremen, Germany) equipped with a nano-ESI source. The samples were redissolved in 10 µL 0.1% FA/H2O and 1 µL was loaded onto the analytical column (Acclaim PepMap C18, 50 µm × 15 cm) and separated using a 60 min gradient. For the gradient separation (mobile phase A, 0.1% FA/H2O; mobile phase B, 0.1% FA/ACN), 3-8% B for 3 min, 8%-28% B for 42 min, 28%-38% B for 9 min, 38%-100% B for 2 min, 100% B for 2 min and finally equilibration with A for 15 min. The flow rate was maintained at 300 nL/min. All MS and MS/MS spectra were acquired in datadependent acquisition mode. The electrospray voltage was set to 1.9 kV and the full MS scan was from m/z 350-1600 with MS resolution of 70 000 and MS/MS resolution of 17 500. The 20 most intense parent ions were fragmented by HCD with NCE of 28% and the AGC target was set to 1×105 with a

maximum injection time of 45 ms. One microscan was recorded using dynamic exclusion of 30 s. Database Search and Data Analysis. Proteome Discoverer software (Thermo Fisher Scientific, version 1.4) with Sequest HT search engine was used for all database search and the database was the Human UniProtKB/Swiss-Prot database (Release 2018-01-26, with 20189 sequences). The data retrieval parameters were as follows: precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.05 Da. Two missed cleavages were allowed by trypsin digestion. False discovery rates (FDR) were controlled lower than 1% by the percolator algorithm at both peptide and protein level. For glycopeptides, carbamidomethyl on cysteine was set as a fixed modification. Oxidation on methionine and deamidation on asparagine were set as variable modifications. Only glycopeptides with the Nglycosylation consensus sequence (N-!P-S/T/C) were considered to be reliable. For phosphopeptides, carbamidomethyl on cysteine was set as fixed modification. Oxidation on methionine and phosphorylation of serine/threonine/tyrosine were set as variable modifications. RESULTS AND DISCUSSION

Synthesis and Characterization of MagG@PEI@PATi4+ Nanocomposites. The synthetic procedure of MagG@PEI@PA-Ti4+ nanocomposites presents in Scheme 1 a. The MagG@PEI nanocomposites were obtained by a onepot synthesis method. Briefly, graphene and PEI were added to the reaction system during the solvothermal synthesis of Fe3O4 nanoparticles. Graphene served as the substrate for Fe3O4 nanoparticles growth while being modified with PEI. Then PA and Ti4+ were coated on the surface of MagG@PEI nanocomposites via electrostatic interactions at room temperature. The morphology of the MagG@PEI@PA-Ti4+ nanocomposites was characterized by SEM and TEM. Figure 1a exhibits the SEM image of the MagG@PEI@PA-Ti4+, showing a graphene lamellar structure with Fe3O4 nanoparticles, which suggests the successful synthesis of magnetic graphene nanocomposites. The lamellar structure of graphene and the particles of Fe3O4 in TEM image (Figure 1b) are consistent with the SEM image. High-resolution TEM (HRTEM) image of the MagG@PEI@PA-Ti4+ (Figure 1c) with the corresponding EDX elemental mapping images (Figure 1d-i) clearly reveal that C, Fe, O, N, P and Ti elements are homogeneously distributed in MagG@PEI@PA-Ti4+ nanocomposites, implying the successful modification of PEI, PA and Ti4+ on the surface of magnetic graphene via the facilely proposed approach. In addition, the EDS spectrum of the MagG@PEI@PA-Ti4+ (Figure 2a) also indicates that C, Fe, O, N, P and Ti are the main elements in MagG@PEI@PA-Ti4+, which is consistent



Scheme 1. (a) Synthetic procedure of MagG@PEI@PATi4+ nanocomposites. (b) Workflow of target peptides enrichment using MagG@PEI@PA-Ti4+ nanocomposites.

Figure 1. Images of SEM (a) and TEM (b) of MagG@PEI@PATi4+ nanocomposites; HRTEM image of MagG@PEI@PA-Ti4+ (c) with the corresponding EDX elemental mapping images of C (d), Fe (e), O (f), N (g), P (h) and Ti (i).

with the results of element mapping. FT-IR was further employed for qualitative characterization (Figure 2b). Compared to MagG@PEI, two new adsorption peaks (1060 cm-1 and 787 cm-1) can be seen in MagG@PEI@PA due to the existence of phosphate group (PO43-), indicating the successful coating of PA on MagG@PEI. Moreover, zeta potential analysis was performed to monitor the changes in surface potential of the MagG@PEI, MagG@PEI@PA and MagG@PEI@PA-Ti4+. As shown in Figure 2c, the zeta potential of the MagG@PEI surface was +51.5 mV, which was attributed to the large amount of amino groups that present in the PEI. After modified with PA, the zeta potential of the MagG@PEI@PA exhibited a negative surface charge due to the six phosphate groups of PA. However, when Ti4+ was immobilized on the surface of MagG@PEI@PA, the zeta potential increased to +4.7 mV. The changes of zeta potential indicate that the MagG@PEI@PA-Ti4+ nanocomposites were successfully synthesized based on the electrostatic interactions. The magnetic properties of the synthetic products were investigated with a vibrating sample magnetometer. The magnetic hysteresis curves reveal that the saturation magnetization (Ms) values reduced slightly after each modification (Figure 2d). Nevertheless, the Ms value of the MagG@PEI@PA-Ti4+ nanocomposites was also sufficient to separate the materials from the solution by using a magnet (as shown in Figure 2d inset). Enrichment of N-Glycopeptides and Phosphopeptides from Standard Proteins. Based on the excellent hydrophilicity endowed by PEI and PA, the MagG@PEI@PA-Ti4+ nanocomposites can capture N-glycopeptides by employing hydrophilic interaction. Therefore, the tryptic digest of human IgG was selected to evaluate the performance of MagG@PEI@PA-Ti4+ for N-glycopeptides enrichment. The workflow of the target peptides enrichment by MagG@PEI@PA-Ti4+ is shown in Scheme 1b. For glycopeptides enrichment with HILIC materials, the enrichment efficiency is primarily based on the ratio of acetonitrile (ACN) to water. In this work, ACN/H2O/TFA (90/8/2, v/v/v) as loading

buffer and H2O/TFA (97/3, v/v) as elution buffer (already be optimized) were applied for glycopeptides enrichment. The MALDI-TOF mass spectra of IgG digest before and after enrichment are shown in Figure 3. No signal of

Figure 2. EDS spectrum (a) of MagG@PEI@PA-Ti4+ nanocomposites; FT-IR spectra (b), Zeta potential (c) and magnetic hysteresis curves (d) of MagG@PEI, MagG@PEI@PA and MagG@PEI@PA-Ti4+ nanocomposites; The inset in (d) shows the dispersion (i) and separation (ii) processes of the MagG@PEI@PA-Ti4+ nanocomposites.

N-glycopeptides could be identified in direct MS analysis (Figure 3a) due to the severe inhibition of a large amount of non-glycopeptides. However, after enrichment by MagG@PEI@PA-Ti4+ (Figure 3b), forty N-glycopeptides (detailed information see Table S1) could be clearly observed with a clean background. The number of the captured Nglycopeptides from IgG digest exceeded those of many previously reported hydrophilic nanomaterials, such as chitosancoated magnetic nanoparticles (twenty-one),34 zwitterionic polymer-coated core-shell magnetic nanoparticles (twentysix) 35 and iminodiacetic acid-functionalized magnetic mesoporous silica (thirty-three) 36. The MagG@PEI@PA-Ti4+ nanocomposites are expected to be one of the most effective materials for N-glycopeptides enrichment. The outstanding enrichment performance of MagG@PEI@PA-Ti4+ toward Nglycopeptides is attributed to the excellent hydrophilicity of PEI and PA. To test the reusability of MagG@PEI@PA-Ti4+ for N-glycopeptides enrichment, the material was recycled by alternate washing with eluent and loading buffer. The regenerated


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Figure 3. MALDI-TOF mass spectra of the tryptic digest of human IgG (2×10-7 M, 200 µL) (a) before and (b) after enrichment by MagG@PEI@PA-Ti4+ nanocomposites. N-Glycopeptides are labeled with “●”.

the MagG@PEI@PA-Ti4+ also has a good enrichment performance for phosphopeptides. Moreover, the MagG@PEI@PATi4+ could also be recycled and reused for phosphopeptides enrichment (Figure S2). Controllable Selective Enrichment of N-Glycopeptides and Phosphopeptides. Glycoproteins and phosphoproteins often coexist in many real biological samples. Encouraged by the excellent enrichment performance of MagG@PEI@PATi4+ toward N-glycopeptides and phosphopeptides, we attempted to use MagG@PEI@PA-Ti4+ to enrich Nglycopeptides and phosphopeptides simultaneously or respectively by changing the enrichment conditions, which was based on the different binding ability of N-glycopeptides and phosphopeptides on the MagG@PEI@PA-Ti4+ nanocomposites. The mixtures of IgG and α-casein digest (molar ratio of 1:1) were used as samples and the schematic diagram of the controllable selective extraction is shown in Scheme 2. Three enrichment conditions were performed in this work: (1) ACN/H2O/TFA=50/48/2 (loading buffer) + 0.4 M NH4OH solution (eluent); (2) ACN/H2O/TFA=90/8/2 (loading buffer) + H2O/TFA=97/3 (eluent 1) + 0.4 M NH4OH solution (eluent 2); (3) ACN/H2O/TFA=90/8/2 (loading buffer) + 0.4 M

Scheme 2. Schematic diagram of the controllable selective enrichment of N-glycopeptides and phosphopeptides from the tryptic digest mixtures of IgG and α-casein using MagG@PEI@PA-Ti4+ nanocomposites.

Figure 4. MALDI-TOF mass spectra of the tryptic digest of αcasein (2×10-7 M, 200 µL) (a) before and (b) after enrichment by MagG@PEI@PA-Ti4+ nanocomposites. Phosphopeptides are labeled with “★”. # indicates dephosphorylated peptides.

MagG@PEI@PA-Ti4+ was reused to enrich N-glycopeptides from IgG tryptic digest for five times. As shown in Figure S1, the MS spectra of N-glycopeptides after being enriched five times were similar to that for the first time, indicating a good reusability of the MagG@PEI@PA-Ti4+. In addition, since the PA molecule carries with six phosphate groups, it can coordinate with Ti4+ to form IMAC material, which indicates that the MagG@PEI@PA-Ti4+ nanocomposites can also be applied for phosphopeptides enrichment. Therefore, α-casein tryptic digest was used as a sample to evaluate the phosphopeptides enrichment performance of MagG@PEI@PA-Ti4+. For phosphopeptides enrichment, ACN/H2O/TFA (50/48/2, v/v/v) and NH4OH solution (0.4 M) were applied as loading buffer and elution buffer, respectively. As shown in Figure 4a, before enrichment, only four phosphopeptides with low signal to noise (S/N) ratio could be observed together with a large number of non-phosphopeptides. After treatment with MagG@PEI@PA-Ti4+, twenty-six phosphopeptides (detailed information see Table S2) were identified with greatly improved S/N ratio (Figure 4b), indicating that

NH4OH solution (eluent). These results are displayed in Figure 5. When using the enrichment condition of (1), Nglycopeptides could not retain on the surface of MagG@PEI@PA-Ti4+ due to the high proportion of water. Therefore, the eluent contained only phosphopeptides, in which 24 phosphopeptides were identified (Figure 5a). However, when the proportion of ACN in loading buffer was increased to 90% (enrichment conditions of (2)and (3)), Nglycopeptides and phosphopeptides could retain on the surface of MagG@PEI@PA-Ti4+ simultaneously. Afterwards, Nglycopeptides were eluted with eluent 1 (H2O/TFA=97/3). At this time, phosphopeptides still retained on the surface of



Figure 6. MALDI-TOF mass spectra of different concentrations of IgG tryptic digest after enrichment with MagG@PEI@PA-Ti4+ nanocomposites. N-Glycopeptides are labeled with “●”.

Figure 5. MALDI-TOF mass spectra of N-glycopeptides and phosphopeptides enriched from the tryptic digest mixtures of IgG and α-casein (molar ratio of 1:1) with different enrichment conditions using MagG@PEI@PA-Ti4+ nanocomposites. NGlycopeptides are labeled with “●”. Phosphopeptides are labeled with “★”.

MagG@PEI@PA-Ti4+ due to the strong acidity of the eluent 1 and then the remaining phosphopeptides were eluted with 0.4 M NH4OH solution (eluent 2). As can be seen from Figure 5b and 5c, 34 N-glycopeptides were observed in eluent 1 and 21 phosphopeptides were observed in eluent 2, indicating that the MagG@PEI@PA-Ti4+ could achieve simultaneous enrichment and separate elution of N-glycopeptides and phosphopeptides under the enrichment condition of (2). Furthermore, the MagG@PEI@PA-Ti4+ could also achieve simultaneous enrichment and simultaneous elution of N-glycopeptides and phosphopeptides under the enrichment condition of (3), which were based on the high proportion of water and the alkalinity of 0.4 M NH4OH solution. As shown in Figure 5d, 32 Nglycopeptides and 25 phosphopeptides were detected simultaneously. It seems to be the largest number of N-glycopeptides and phosphopeptides to be identified simultaneously in one mass spectrum. These results indicate that the MagG@PEI@PA-Ti4+ can be used to enrich N-glycopeptides and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glycopeptides and phosphopeptides. Evaluation of N-Glycopeptides and Phosphopeptides Enrichment Performance. In order to further demonstrate the excellent enrichment performance of MagG@PEI@PA-Ti4+, the selectivity and sensitivity of MagG@PEI@PA-Ti4+ toward N-glycopeptides and phosphopeptides were also investigated. For selectivity evaluation, the tryptic digest mixtures of glycoprotein (IgG)/phosphoprotein (α-casein) and BSA with different molar ratios were used to simulate complex samples. The direct analysis of the peptide mixtures of IgG and BSA with a 1:100 molar ratio is shown in Figure S3a, non-glycopeptides dominated the spectrum and no glycopeptides were observed. However, after treatment with MagG@PEI@PA-Ti4+, almost all of the non-target peptides were removed and thirty-five


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Figure 8. Evaluation of the loading capacity of MagG@PEI@PA-Ti4+ for (a) N-glycopeptides and (b) phosphopeptides with three replicates.

Figure 7. MALDI-TOF mass spectra of different concentrations of α-casein tryptic digest after enrichment with MagG@PEI@PATi4+ nanocomposites. Phosphopeptides are labeled with “★”.

N-glycopeptides were identified with significantly enhanced signal intensity (Figure S3b). Continuing to increase the molar ratio to 1:1000, fifteen N-glycopeptides could still be observed (Figure S3d). For HILIC-based materials, it is a very excellent selectivity for N-glycopeptides enrichment. Similarly, for phosphopeptides, the digest mixtures of α-casein and BSA with different molar ratios (1:500, 1:1000 and 1:5000) were enriched by MagG@PEI@PA-Ti4+. Before enrichment, the signals of phosphopeptides were completely suppressed by abundant non-phosphopeptides when the molar ratio of αcasein and BSA was 1:500 (Figure S4a). However, after enrichment, seventeen phosphopeptides were observed with significantly improved signal intensity (Figure S4b). Even at a molar ratio of 1:5000 (Figure S4d), thirteen phosphopeptides still dominated the spectrum, indicating an excellent selectivity of MagG@PEI@PA-Ti4+ for phosphopeptides enrichment. For sensitivity measurement, different concentrations of IgG digest (2 fmol/µL, 1 fmol/µL and 0.5 fmol/µL) / α-casein digest (0.5 fmol/µL, 0.2 fmol/µL and 0.1 fmol/µL) were used as samples for testing. Figure 6 reveal that even when the concentration of IgG digest was as low as 0.5 fmol/µL, three Nglycopeptides were still identified with the S/N ratio over 3. And for phosphopeptides, the minimum detection limit of MagG@PEI@PA-Ti4+ was as low as 0.1 fmol/µL (Figure 7). Obviously, the MagG@PEI@PA-Ti4+ nanocomposites possess excellent selectivity and sensitivity for Nglycopeptides/phosphopeptides enrichment, indicating that the MagG@PEI@PA-Ti4+ can be used to capture Nglycopeptides/phosphopeptides even if the sample is extremely complex and the N-glycopeptides/phosphopeptides concentration is extremely low. The enrichment capacity of the MagG@PEI@PA-Ti4+ toward N-glycopeptides and phosphopeptides was also investigated by adding different amounts of nanocomposites to a

fixed amount of IgG (6 µg) and α-casein tryptic digest (1 µg), respectively. As shown in Figure 8, it can be calculated that the loading capacity is up to about 300 mg g-1 for Nglycopeptides and 100 mg g-1 for phosphopeptides. Furthermore, IgG tryptic digest (3 µg) and β-casein tryptic digest (1 µg) were respectively used to evaluate the recovery of Nglycopeptides and phosphopeptides after enrichment with MagG@PEI@PA-Ti4+, by using the quantitative approach of stable isotope dimethyl labeling (Figure S5 and Figure S6). As shown in Table S3 and Table S4, the recovery of MagG@PEI@PA-Ti4+ was over 90% for both Nglycopeptides and phosphopeptides enrichment, which was higher than that of many previous reports.37-40 Identification of N-Glycopeptides and Phosphopeptides from HeLa Cell Extracts. In order to further examine the effectiveness of MagG@PEI@PA-Ti4+ nanocomposites for the specific enrichment of low-abundant N-glycopeptides and phosphopeptides from a real complex sample, HeLa cell extracts containing both N-glycopeptides and phosphopeptides were selected as a sample. A 200 µg amount of proteins extracted from HeLa cells and digested with trypsin was enriched by the MagG@PEI@PA-Ti4+ nanocomposites. In this process, ACN/H2O/TFA (90/8/2, v/v/v) was used as loading buffer to ensure that both N-glycopeptides and phosphopeptides remained on the surface of MagG@PEI@PA-Ti4+. Subsequently, the N-glycopeptides were eluted with 3% TFA aqueous solution and deglycosylated with PNGase F, and then the remaining phosphopeptides were eluted with 0.4 M NH4OH solution. The eluents were analyzed by nano-LCMS/MS, respectively. The identification of N-glycopeptides was confirmed by the existence of a consensus sequence (N!P-S/T/C) and deamidation on the asparagines residues with a mass increment of 0.9858 Da. A total of 393 N-glycopeptides derived from 259 glycoproteins and 574 phosphopeptides derived from 341 phosphoproteins (Table S5 and Table S6) were finally identified in two independent LC-MS/MS runs (60 min per run). This result reveals that the MagG@PEI@PA-Ti4+ can simultaneously enrich N-glycopeptides and phosphopeptides from real biological samples in a simple manner with high efficiency. CONCLUSION In conclusion, novel nanocomposites MagG@PEI@PA-Ti4+, with PA as a new chelating ligand, were successfully synthesized by a facile method. The MagG@PEI@PA-Ti4+ nanocomposites possessed combined properties of IMAC- and HILIC-based materials with large surface area, excellent hydrophilicity and high Ti4+ loading amount. Due to the large amount of Ti4+ and excellent hydrophilicity of PEI and PA, MagG@PEI@PA-Ti4+ can be used for N-glycopeptides and phosphopeptides enrichment with high selectivity and high detection sensitivity. According to the different binding ability



of MagG@PEI@PA-Ti4+ toward N-glycopeptides and phosphopeptides, the MagG@PEI@PA-Ti4+ can enrich Nglycopeptides and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glycopeptides and phosphopeptides. In addition, the MagG@PEI@PA-Ti4+ nanocomposites can simultaneously enrich N-glycopeptides and phosphopeptides from HeLa cell extracts with high efficiency. Due to the stronger binding ability of MagG@PEI@PA-Ti4+ to phosphopeptides than Nglycopeptides, the N-glycopeptides on the nanocomposites can be first eluted with 3% TFA aqueous solution, and the remaining phosphopeptides were eluted with 0.4 M NH4OH solution for two independent LC-MS/MS analysis. All in all, the MagG@PEI@PA-Ti4+ nanocomposites have great advantages in identifying low abundance N-glycopeptides and phosphopeptides, even from real biological samples. We believe that the MagG@PEI@PA-Ti4+ will have high application potential in N-glycoproteome and phosphoproteome research.

ASSOCIATED CONTENT Supporting Information Detailed information of N-glycopeptides obtained from IgG tryptic digest; Detailed information of phosphopeptides obtained from α-casein tryptic digest; MALDI mass spectra of the reusability, enrichment selectivity and recovery for N-glycopeptides and phosphopeptides enrichment; Detailed information of Nglycopeptides and phosphopeptides obtained from HeLa cell extracts.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (M. B. L) * E-mail: [email protected] (H. L. Z)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the Science and Technology Commission of Shanghai Municipality (STCSM, No. 16520710800) and the Fundamental Research Funds for the Central Universities (No. 222201817022).

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