- Email: [email protected]
phosphopeptides
Talanta 197 (2019) 77–85
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Hydrophilic phytic acid-functionalized magnetic dendritic mesoporous silica nanospheres with immobilized Ti4+: A dual-purpose affinity material for highly efficient enrichment of glycopeptides/phosphopeptides Yayun Honga,1, Qiliang Zhana,1, Yu Zhenga, Chenlu Pua, Hongli Zhaoa, , Minbo Lana,b, ⁎
T
⁎⁎
a
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 b State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Magnetic mesoporous silica microspheres Phytic acid LbL assembly N-glycopeptides enrichment Phosphopeptide enrichment
In this work, magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels were fabricated by a self-assembly synthesis in chlorobenzene-water mixed system. Then, the obtained Mag-MSMs were modified with polyethyleneimine (PEI), phytic acid (PA) and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ possessed combined properties of hydrophilic interaction liquid chromatography (HILIC)- and immobilized metal ion affinity chromatography (IMAC)-based materials, which could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The proposed Mag-MSMs@ PEI-PA-Ti4+ exhibited an outstanding performance for N-glycopeptides enrichment (selectivity: IgG/ BSA = 1:1000; sensitivity: 0.5 fmol/μL IgG) and phosphopeptides enrichment (selectivity: α-casein/ BSA=1:5000; sensitivity: 0.2 fmol/μL α-casein). Furthermore, after enrichment with Mag-MSMs@PEI-PA-Ti4+, a total of 276 N-glycopeptides assigned to 132 glycoproteins were identified from 2 μL human serum and 1645 phosphopeptides corresponding to 704 phosphoproteins were identified from 200 μg HeLa cell extracts.
1. Introduction Protein post-translational modifications (PTMs), especially glycosylation and phosphorylation, play a key role in many important biological processes and often participate in the development of various diseases [1–3]. Abnormal glycoprotein and phosphoprotein with high clinical sensitivity and specificity can be used as disease biomarkers for clinical diagnosis [4–7]. Currently, mass spectrometry (MS) is the most powerful platform for protein PTMs research to accurately identify and locate glycosylation/phosphorylation sites [8–10]. However, it is still difficult to study these two PTMs directly with MS due to the severe suppression caused by the coexistence of high abundant unmodified peptides [11,12]. Therefore, an effective enrichment prior to MS analysis is a prerequisite for accurate identification of glycopeptides/ phosphopeptides from biological samples. To date, a lot of affinity materials are available for peptides enrichment, especially nanomaterials have been widely used in
biomolecule enrichment due to their large surface area and easy postfunctionalization [13–15]. Nanomaterials, such as silica [16,17], graphene [18–20] and metal-organic frameworks (MOFs) [21–23] are commonly used as substrates for the synthesis of affinity materials for peptides enrichment. Among them, mesoporous silica has attracted increasing attention in the field of adsorption separation due to its tunable structures and morphologies. However, conventional mesoporous silica with worm-like mesochannels have relatively small pore sizes (< 2.5 nm) [24,25], which will hamper the subsequent modification of the mesopores walls. In this regard, mesoporous silica with ordered radial mesochannels is considered as an alternative. The unique radial mesochannels can provide a large and easily accessible mesopore, which can facilitate the subsequent modification. Therefore, it is of great importance to develop a facile method to synthesize mesoporous silica microspheres with ordered radial mesochannels. Currently, based on the hydrophilicity of glycopeptides, hydrophilic interaction liquid chromatography (HILIC) has matured to enrich N-
Corresponding author. Corresponding author at: 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. E-mail addresses: [email protected] (H. Zhao), [email protected] (M. Lan). 1 Author contributes equally to this work. ⁎
⁎⁎
https://doi.org/10.1016/j.talanta.2019.01.005 Received 29 October 2018; Received in revised form 26 December 2018; Accepted 2 January 2019 Available online 03 January 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 197 (2019) 77–85
Y. Hong et al.
glycopeptides through hydrophilic interaction [26,27]. And based on the reversible affinity between phosphate groups and immobilized metal ions, immobilized metal ion affinity chromatography (IMAC) has been widely applied to phosphopeptide enrichment [28,29]. However, a lot of affinity materials can only be used for separate enrichment of glycopeptides or phosphopeptides. When studying glycopeptides and phosphopeptides simultaneously, two different materials should be prepared, one for glycopeptides enrichment and the other for phosphopeptides enrichment. Thus, dual-purpose materials with combined properties of HILIC and IMAC for N-glycopeptides and phosphopeptides enrichment have been paid much attention [30–32]. Phytic acid (PA), an eco-friendly and renewable compound, is considered as an excellent modification material. On one hand, PA with six phosphate groups can offer strong affinity sites for metal ions binding to form an IMAC affinity material [33–35]. On the other hand, the excellent hydrophilicity of PA can endow affinity material with HILIC performance [34,35]. Therefore, the affinity material modified with PA can be used for both phosphopeptides and glycopeptides enrichment. Herein, we fabricated magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels through a self-assembly synthesis in chlorobenzene-water mixed system, then the Mag-MSMs were modified with polyethyleneimine (PEI), PA and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. The unique radial pore structure facilitated more PA grafting onto the pore walls of mesoporous silica shell, thereby increasing the binding amount of immobilized Ti4+. Due to the excellent hydrophilicity of PEI and PA and a large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The selectivity and sensitivity of the Mag-MSMs@PEIPA-Ti4+ in N-glycopeptides and phosphopeptides enrichment have been evaluated by using human immunoglobulin G (IgG) digest and αcasein digest, respectively. Furthermore, the Mag-MSMs@PEI-PA-Ti4+ also showed a great advantage in capturing N-glycopeptides from human serum and identifying phosphopeptides from HeLa cell extracts, indicating its great potential in protein PTMs research.
Fe3O4@RF in a chlorobenzene-water system. Briefly, 0.1 g Fe3O4@RF was dispersed in 250 mL deionized water by ultrasonication. Then, 0.68 g of TEA was added and the mixture was mechanically stirred at 80 °C for 0.5 h. Afterwards, 1.52 g CTAB were added and kept under stirring for 1 h, followed by the addition of the mixture of 15 mL of chlorobenzene and 100 μL of APTES. The reaction solution was kept under stirring at 80 °C for another 1 h. Finally, 3 mL TEOS were added in the reaction solution and stirred for 24 h. The products were collected with a magnet and washed alternately with ethanol (three times) and deionized water (three times), followed by extraction with ethanol at 65 °C for three times to remove the template and dried in vacuum at 45 °C overnight. 2.3. Synthesis of Mag-MSMs@PEI-PA-Ti4+ PEI, PA and Ti4+ were modified onto the surface of Mag-MSMs via LbL assembly. The dried Mag-MSMs were dispersed in 100 mL PEI aqueous solution (5 mg/mL) by 5 min ultrasonication and stirred for 12 h at room temperature. The obtained nanoparticles were collected by magnetic separation and the excess PEI was removed by three washings with deionized water. After that, Mag-MSMs@PEI were then redispersed in 100 mL PA aqueous solution (8.4 mg/mL) and stirred for 6 h at room temperature, magnetic separation and followed by three washings. Finally, the Mag-MSMs@PEI-PA were incubated in 50 mL Ti (SO4)2 solution (100 mM) for 2 h to immobilize Ti4+. The Mag-MSMs@ PEI-PA-Ti4+ were collected with a magnet and rinsed with deionized water for three times, followed by lyophilization to dryness. 2.4. Characterization of Mag-MSMs@PEI-PA-Ti4+ Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1400 transmission electron microscope (JEOL, Japan). Energy dispersive spectroscopy (EDS) images were conducted on Falion 60S (EDAX, USA). Zeta potential measurements were performed on a Nano-ZS90 instrument (Malvern, UK) in water at 25 °C. The magnetic hysteresis curves were carried out on a Lakeshore 7407 vibrating sample magnetometer (Lakeshore, USA) at room temperature. Nitrogen adsorption-desorption isotherms were measured on an ASAP 2020 apparatus (Micrometritics, USA). Fourier transform infrared (FTIR) spectra were obtained from Nicolet 6700 (Thermo Fisher Scientific, USA) using KBr pellets.
2. Experimental 2.1. Materials and chemicals Triethanolamine (TEA), cetyltrimethylammonium bromide (CTAB), chlorobenzene, 3-aminopropyltriethoxysilane (APTES), tetraethylorthosilicate (TEOS), polyethyleneimine (PEI, 50%, Mw=70000), phytic acid (PA, 70%) and Ti(SO4)2 were purchased from Aladdin (Shanghai, China). Bovine serum albumin (BSA), α-casein (from bovine milk), immunoglobulin G (IgG) from human serum, ammonium bicarbonate (NH4HCO3), urea, dithiothreitol (DTT), iodoacetamide (IAA), trypsin and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptide-N-glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA, USA). Acetonitrile (ACN), trifluoroacetic acid (TFA) and formic acid (FA) were of chromatographic grade. Human serum samples were collected from healthy people and obtained from the Sixth People's Hospital affiliated to Shanghai Jiaotong University according to the standard clinical procedures. Ultrapure water (18.2 MΩ cm) was produced using a Milli-Q system (Millipore, Bedford, MA, USA). All chemicals were used as received without purifcation.
IgG, BSA and α-casein were dissolved in 50 mM NH4HCO3 buffer (pH 8.2) and treated with trypsin (enzyme to protein ratio of 1:25, w/ w) at 37 °C for 17 h for digestion. IgG and BSA were reduced by DTT and alkylated by IAA. α-Casein was digested directly. For preparation of human serum digest, 2 μL of human serum were diluted with 18 μL NH4HCO3 buffer (50 mM, pH 8.2) containing 8 M urea and reduced by DTT at 56 °C for 45 min, and then alkylated by IAA in dark for 30 min. Finally, the solution was diluted to 50 μL with NH4HCO3 buffer and treated with trypsin at 37 °C for 17 h at an enzyme to protein ratio of 1:25 (w/w). Tryptic digest was lyophilized for further use. The proteins from HeLa cells were extracted as previously described [36] and the process of protein digestion was the same as that of IgG.
2.2. Synthesis of Mag-MSMs
2.6. N-glycopeptides and phosphopeptides enrichment
Magnetic Fe3O4 nanoparticles were synthesized by a solvothermal method and then coated with resorcinol-formaldehyde (RF) resin to form magnetic RF resin microspheres (denoted as Fe3O4@RF) through interface sol-gel polymerization of resorcinol and formaldehyde as described by Yue et al. [25]. Subsequently, the mesoporous silica shell with ordered radial mesochannels were coated on the surface of
For glycopeptides enrichment from IgG digest, 20 μg of MagMSMs@PEI-PA-Ti4+ was dispersed in 200 μL of loading buffer (ACN/ H2O/TFA=90/8/2, v/v/v) containing 2 × 10−7 M IgG digest and then incubated at room temperature for 30 min. After that, the microspheres 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,
2.5. Sample preparation
78
Talanta 197 (2019) 77–85
Y. Hong et al.
Scheme 1. Synthetic procedure of Mag-MSMs@PEI-PA-Ti4+.
the captured glycopeptides were eluted with 10 μL 3% TFA aqueous solution for 15 min. The eluent was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For glycopeptides enrichment from human serum digest, 2 μL of the lyophilized serum digest was redissolved in 500 μL of loading buffer (ACN/H2O/TFA = 90/8/2, v/v/v) and then incubated with 400 μg of Mag-MSMs@PEI-PA-Ti4+ for 30 min. Subsequently, the microspheres 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 with 3% TFA aqueous solution (100 μL × 3) and then lyophilized for deglycosylation. The lyophilized glycopeptides were redissolved in 19 μL of 50 mM NH4HCO3 and 1 μL (500 unites) of PNGaseF was added. The mixture was incubated at 37 °C for 16 h and then lyophilized for nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis. For phosphopeptides enrichment from α-casein digest, 20 μg of MagMSMs@PEI-PA-Ti4+ was dispersed in 200 μL of loading buffer (ACN/ H2O/TFA=50/48/2, v/v/v) containing 2 × 10−7 M α-casein digest and then incubated at room temperature for 30 min. After that, the microspheres were collected 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 phosphopeptides enrichment from HeLa cell extracts, 200 μg of tryptic digests were diluted with 500 μL of loading buffer (ACN/H2O/ TFA = 70/29/1, v/v/v) and incubated with 2 mg of Mag-MSMs@PEIPA-Ti4+ for 30 min. Subsequently, the microspheres were collected and rinsed with 500 μL of washing buffer (ACN/H2O/TFA = 70/29.9/0.1, v/v/v) for three times. The captured phosphopeptides were eluted with 0.4 M NH4OH solution (100 μL × 3) and then lyophilized for nano-LCMS/MS analysis directly.
2.7. Mass spectrometry analysis MALDI-TOF MS experiments were carried out in positive ion mode by a 4800 Plus MALDI-TOF MS (AB Sciex, USA) with a Nd:YAG laser emitting at 355 nm. 1 μL analyte was dropped on the plate, then 1 μL matrix (DHB, 25 mg/mL, ACN/H2O/H3PO4 = 70:29:1, v/v/v) was dropped and analyzed by MALDI-TOF MS. Nano-LC-MS/MS experiments were performed using an EASY-nLC 1000 Nano HPLC System (Thermo Fisher Scientific, Bremen, Germany) with a quadrupole-Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Bremen, Germany). The lyophilized 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), then separated using a 60 min gradient. For reversed phase liquid chromatographic (RPLC) separation, 0.1% FA/H2O and 0.1% FA/ACN were used as the mobile phase A and B, respectively. The reversed phase (RP) gradient elution was performed as follows: 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 10 min. The flow rate was maintained at 300 nL/min. All MS and MS/MS spectra were acquired in data-dependent 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 higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28% and the automatic gain control (AGC) target was set to 3 × 105 with a maximum injection time of 45 ms. Dynamic exclusion (30 s) was on. 2.8. Database search and data analysis The MS raw data files were searched by Proteome Discoverer software (Thermo Fisher Scientific, version 1.4) with Sequest HT search 79
Talanta 197 (2019) 77–85
Y. Hong et al.
Ti4+ were confirmed through diverse characterizations. TEM images of the products obtained after each synthesis step are shown in Fig. 1. The water dispersible Fe3O4 nanoparticles synthesized by a solvothermal method possess a uniform size of 250 nm without aggregation (Fig. 1a). After RF resin coating, a RF resin layer with thickness of ~30 nm can be clearly observed on Fe3O4@RF nanoparticles (Fig. 1b), indicating that the RF resin has been successfully modified on Fe3O4 nanoparticles through the interface sol-gel polymerization of resorcinol and formaldehyde. Subsequently, the mesoporous silica shell was formed on Fe3O4@RF nanoparticles in a chlorobenzene-water mixed system. After removal of CTAB through extraction, the Mag-MSMs exhibit distinct core-shell-shell structure with an ordered radial mesochannels (Fig. 1c). With the modification of PEI, PA and Ti4+, the mesoporous channels gradually decreased, indicating that PEI, PA and Ti4+ have been successfully grafted onto the pore walls of mesoporous silica shell via LbL assembly (Fig. 1d–f). The stepwise coating of PEI, PA and Ti4+ on the Mag-MSMs was monitored by observing zeta potential changes of Mag-MSMs after each modification step. As shown in Fig. 2a, the zeta potential of the MagMSMs was −21.9 mV due to the presence of a large number of silicon hydroxyl. After PEI coating, the zeta potential changed from negative to positive, which was attributed to the numerous amino groups presented in PEI. Subsequently, grafting with PA, the zeta potential of the microspheres changed negative again due to the six phosphate groups of PA. Finally, after Ti4+ chelating, the zeta potential of the Mag-MSMs@ PEI-PA-Ti4+ increased to +16.9 mV. The changes of zeta potential indicate the successful introduction of functional group (amino groups, phosphate group and titanium ion) on Mag-MSMs via LbL procedure. In addition, the EDS spectrum (Fig. 2b) and FT-IR spectra (Fig. S1) further confirmed the successful modification on Mag-MSMs. The N2 adsorption-desorption analysis was performed to study the porosity of MagMSMs@PEI-PA-Ti4+ (Fig. 2c). The pore size distribution curve reveals an average pore size of 3.6 nm (Fig. 2c inset), which is suitable for low
engine and the database was the Human UniProtKB/Swiss-Prot database (Release 2018-01-26, with 20189 sequences). Two missed cleavages were allowed by trypsin digestion. The mass tolerances for the precursor ions and fragment ions were set to 10 ppm and 0.05 Da, respectively. False discovery rates (FDR) were set to 1% for both peptide and protein. Carbamidomethyl on cysteine (C) was chosen for fixed modifications, and oxidation on methionine (M) was set as variable modifications. For phosphopeptides, phosphorylation of serine/threonine/tyrosine (S/T/Y) were set as variable modifications. For glycopeptides, deamidation on asparagine (N) was set as variable modifications. Only glycopeptides with the N-glycosylation consensus sequence (N-! Proline-S/T/C) were considered to be reliable. 3. Results and discussion 3.1. Synthesis and characterization of Mag-MSMs@PEI-PA-Ti4+ The synthetic procedure of Mag-MSMs@PEI-PA-Ti4+ is illustrated in Scheme 1. The Mag-MSMs were prepared in a chlorobenzene-water mixed system, using Fe3O4@RF nanoparticles as seeds, TEA as a catalyst, CTAB as a surfactant, TEOS and APTES as silica sources and chlorobenzene as swelling agents. Firstly, a water phase containing Fe3O4@RF nanoparticles, CTAB and TEA was mixed with an oil phase containing APTES and chlorobenzene, then TEOS was added into the mixture. During the reaction process, negatively charged silica oligomers derived from TEOS hydrolysis coassembled with positively charged CTAB micelles and protonated APTES into rod-like composites through electrostatic interaction, and deposited on Fe3O4@RF nanoparticles to form Mag-MSMs [25,37]. The Mag-MSMs were collected with a magnet followed by surfactant extraction in ethanol. Then, PEI, PA and Ti4+ were grafted onto the inwalls of mesoporous channels via LbL assembly at room temperature to obtain Mag-MSMs@PEI-PA-Ti4+. The morphology and surface modification of Mag-MSMs@PEI-PA-
Fig. 1. TEM images of (a) Fe3O4, (b) Fe3O4@RF, (c) Mag-MSMs, (d) Mag-MSMs@PEI, (e) Mag-MSMs@PEI-PA and (f) Mag-MSMs@PEI-PA-Ti4+. 80
Talanta 197 (2019) 77–85
Y. Hong et al.
Fig. 2. (a) Zeta potential changes of microspheres during LbL assembly procedure; (b) EDS spectrum of Mag-MSMs@PEI-PA-Ti4+; (c) N2 adsorption-desorption isotherm and pore size distribution (inset) of Mag-MSMs@PEI-PA-Ti4+; (d) Magnetic hysteresis curves of Fe3O4 and Mag-MSMs@PEI-PA-Ti4+; Inset: magnetic response of Mag-MSMs@PEI-PA-Ti4+ to an external magnet.
molecular weight peptides enrichment. Moreover, the magnetic property of Mag-MSMs@PEI-PA-Ti4+ were also investigated. Fig. 2d shows that the saturation magnetization value of Mag-MSMs@PEI-PA-Ti4+ was measured to be 18.2 emu/g. Although this value was significantly reduced in comparison to that of original Fe3O4 nanoparticles (62.0 emu/g) due to multilayered modification, the Mag-MSMs@PEIPA-Ti4+ could still be quickly separated by using a magnet (as shown in Fig. 2d inset).
by the large amount of non-glycopeptides and high salt content (Fig. 3a). However, after treatment with Mag-MSMs@PEI-PA-Ti4+, a total of thirty-six N-glycopeptides (detailed information see Table S1) were observed with significantly increased signal intensity (Fig. 3b). The number of the N-glycopeptides identified from human IgG digest exceeded those of many previous HILIC nanomaterials [38–40], which is mainly attributed to the excellent hydrophilicity from the numerous amino groups of PEI and phosphate groups of PA. In addition, the selectivity and sensitivity of Mag-MSMs@PEI-PATi4+ for N-glycopeptides enrichment were also investigated. For selectivity evaluation, BSA, an unmodified protein, was digested by trypsin and then doped into IgG digest with different molar ratios to simulate complex samples. As shown in Fig. 4a, direct analysis of IgG and BSA digest mixture with a molar ratio of 1:100, the spectrum was completely occupied by a large amount of high-intensity non-glycopeptides. While after enrichment with Mag-MSMs@PEI-PA-Ti4+, thirtythree N-glycopeptides with high MS intensity were identified in a clean background. Compared with the number of N-glycopeptides identified from standard IgG digest, there is only a slight decrease in the number after 100-fold BSA doping. With the molar ratio of IgG and BSA increased to 1:1000, twenty-two N-glycopeptides with high signal intensity still occupied the spectrum. Compared with previous HILIC materials, such as MIL-101(NH2)@Au-Cys (IgG:BSA = 1:50, identified 11 N-glycopeptides) [41], Fe3O4@TpPa-1 (IgG:BSA = 1:100, identified 8 N-glycopeptides) [42] and GO-Fe3O4/SiO2/AuNWs/L-Cys (IgG:BSA = 1:100, identified 15 N-glycopeptides) [43], Mag-MSMs@PEIPA-Ti4+ indeed exhibit an excellent selectivity for N-glycopeptides enrichment. Furthermore, the detection sensitivity of Mag-MSMs@PEIPA-Ti4+ was also evaluated by different concentrations of IgG digest (2
3.2. Evaluation of the N-glycopeptides/phosphopeptides enrichment performance of Mag-MSMs@PEI-PA-Ti4+ using standard proteins Based on the excellent hydrophilicity endowed by PEI and PA, MagMSMs@PEI-PA-Ti4+ can be used as a HILIC material for N-glycopeptides enrichment through the hydrophilic interaction between the glycan chains of N-glycopeptides and the material. Simultaneously, due to the large amount of Ti4+ chelated by PA, Mag-MSMs@PEI-PA-Ti4+, also as an IMAC material, was anticipated to have excellent performance for phosphopeptides enrichment. Therefore, tryptic digests of standard glycoprotein (human IgG) and phosphoprotein (α-casein) were employed as the corresponding testing samples for N-glycopeptides/phosphopeptides enrichment. A typical procedure with respective enrichment conditions to enrich N-glycopeptides/phosphopeptides from protein tryptic digests is displayed in Scheme 2, including incubation for peptides adsorption, removing nontarget peptides and elution of N-glycopeptides/phosphopeptides. Fig. 3 shows the enrichment results of N-glycopeptides before and after treatment with MagMSMs@PEI-PA-Ti4+. It can be seen that, before enrichment, no Nglycopeptides could be observed due to the severe suppression caused 81
Talanta 197 (2019) 77–85
Y. Hong et al.
Scheme 2. Workflow of target peptides enrichment from protein tryptic digests using Mag-MSMs@PEI-PA-Ti4+.
Fig. 3. MALDI-TOF mass spectra of human IgG digest (a) before and (b) after enrichment by Mag-MSMs@PEI-PA-Ti4+.
Fig. 5. MALDI-TOF mass spectra of human IgG tryptic digest with different concentrations after enrichment with Mag-MSMs@PEI-PA-Ti4+.
fmol/μL, 1 fmol/μL and 0.5 fmol/μL). As shown in Fig. 5a, thirteen Nglycopeptides were easily detected from 2 fmol/μL of IgG digest after enrichment by Mag-MSMs@PEI-PA-Ti4+. Even when the concentration of IgG digest was reduced to 0.5 fmol/μL, six N-glycopeptides could still be clearly observed. The outstanding enrichment selectivity and sensitivity of Mag-MSMs@PEI-PA-Ti4+ are mainly attributed to the excellent hydrophilicity of PEI and PA. These results reveal that the Mag-MSMs@ PEI-PA-Ti4+ can be used as a HILIC material for efficient N-glycopeptides enrichment from complex biological sample, even at low concentration. Since the PA molecule carries with six phosphate groups, it can further serve as chelating ligands for Ti4+ immobilization to form IMAC material. Based on the reversible specific affinity of the Ti4+ to the phosphate groups of phosphopeptides, Mag-MSMs@PEI-PA-Ti4+ are also suitable for phosphopeptides enrichment. The enrichment performance of Mag-MSMs@PEI-PA-Ti4+ for phosphopeptides was investigated by capturing phosphopeptides from α-casein digest. As shown in Fig. 6a, without enrichment, only four phosphopeptides with low signal intensity were observed together with a large number of non-
Fig. 4. MALDI-TOF mass spectra of the tryptic digest mixtures of human IgG and BSA without enrichment at a molar ratio of 1:100 (a), and with the MagMSMs@PEI-PA-Ti4+ enrichment at molar ratios of 1:100 (b) and 1:1000 (c). 82
Talanta 197 (2019) 77–85
Y. Hong et al.
Fig. 6. MALDI-TOF mass spectra of α-casein digest (a) before and (b) after enrichment by Mag-MSMs@PEI-PA-Ti4+. # indicates dephosphorylated peptides.
phosphopeptides. However, after enrichment with Mag-MSMs@PEI-PATi4+, non-phosphopeptides were entirely removed and thirty phosphopeptides (detailed information see Table S2) were identified with greatly improved signals (Fig. 6b), indicating that the Mag-MSMs@PEIPA-Ti4+ could also be used as an IMAC material for phosphopeptides enrichment. The high enrichment efficiency of Mag-MSMs@PEI-PATi4+ toward phosphopeptides is attributed to the large amount of Ti4+ immobilized by PA via strong chelating interaction. To further explore the selectivity of Mag-MSMs@PEI-PA-Ti4+ for phosphopeptides enrichment, α-casein digest doped with different molar ratios of BSA digest was used as samples. For comparison, the direct analysis of α-casein digest mixed with 1000-fold BSA digest is shown in Fig. 7a. Obviously, no phosphopeptides could be observed except a large number of non-phosphopeptides. However, after
Fig. 8. MALDI-TOF mass spectra of α-casein tryptic digest with the concentrations of 1 fmol/μL (a), 0.5 fmol/μL (b) and 0.2 fmol/μL (c) after enrichment with Mag-MSMs@PEI-PA-Ti4+.
enrichment, twenty-four phosphopeptides completely occupied the MS spectrum (Fig. 7b). Even increasing the amount of BSA digest to 5000fold (Fig. 7c), nineteen phosphopeptides could still be clearly observed, indicating an excellent phosphopeptides enrichment selectivity of MagMSMs@PEI-PA-Ti4+. For sensitivity measurement, Mag-MSMs@PEIPA-Ti4+ were applied to capture phosphopeptides from different concentrations of α-casein digest (1 fmol/μL, 0.5 fmol/μL and 0.2 fmol/μL). As shown in Fig. 8a, eight phosphopeptides were identified in 1 fmol/μL α-casein digest. Even when the concentration of α-casein digest was reduced to 0.2 fmol/μL, two phosphopeptides could still be identified with signal to noise ratio over 3 (Fig. 8c). These results reveal that the Mag-MSMs@PEI-PA-Ti4+ can also be used for phosphopeptides enrichment from complex and low-concentration samples. 3.3. Evaluation of the N-glycopeptides/phosphopeptides enrichment efficiency of Mag-MSMs@PEI-PA-Ti4+ using real biological samples As a common clinical specimen, human serum contains abundant glycoprotein biomarkers, which is of great importance for potential disease diagnosis. Effective enrichment and identification of glycopeptides in human serum is a prerequisite for discovering new disease biomarkers. Therefore, peptides mixture of 2 μL healthy human serum treated with trypsin was selected to examine the N-glycopeptides enrichment efficiency of Mag-MSMs@PEI-PA-Ti4+. The enrichment process was profiled in Scheme 2 by using ACN/H2O/TFA (90/8/2, v/v/v) as loading buffer and 3% TFA aqueous solution as eluent. The eluted Nglycopeptide was deglycosylated with PNGase F and then analyzed by nano-LC-MS/MS. The N-glycopeptides with a consensus sequence (N!P-S/T/C) and the deamidation simultaneously occurred on asparagines residues (mass increment, 0.9858 Da) were considered to be reliable. A total of 276 N-glycopeptides derived from 132 glycoproteins were identified from only 2 μL human serum (detailed information see Table S3). The Mag-MSMs@PEI-PA-Ti4+ shows a higher enrichment efficiency than those of previous publications, such as MIL-101(Cr)-maltose (5 μL human serum: 111 glycopeptides, 65 glycoproteins) [23], Fe3O4@G6P microspheres (2 μL human serum: 243 glycopeptides, 92
Fig. 7. MALDI-TOF mass spectra of the tryptic digest mixtures of α-casein and BSA without enrichment at a molar ratio of 1:1000 (a), and with the MagMSMs@PEI-PA-Ti4+ enrichment at molar ratios of 1:1000 (b) and 1:5000 (c). # indicates dephosphorylated peptides. 83
Talanta 197 (2019) 77–85
Y. Hong et al.
glycoproteins) [44] and UiO-66-COOH (2 μL human serum: 255 glycopeptides, 93 glycoproteins) [45]. In addition, for evaluating the enrichment efficiency of MagMSMs@PEI-PA-Ti4+ toward phosphopeptides, HeLa cell extracts were selected as a real biological sample. In this process, 200 μg HeLa cells extracts were enriched by Mag-MSMs@PEI-PA-Ti4+ with ACN/H2O/ TFA (70/29/1, v/v/v) as loading buffer and 0.4 M NH4OH solution as eluent. The obtained phosphopeptides were lyophilized and then analyzed by nano-LC-MS/MS. A total of 1645 phosphopeptides derived from 704 phosphoproteins were finally identified (detailed information see Table S4). Compared with the commercial TiO2 (1266 phosphopeptides derived from 554 phosphoproteins, the result can be found in our previous work [46]), the Mag-MSMs@PEI-PA-Ti4+ has obvious advantages in phosphopeptides enrichment. These enrichment results indicate that the Mag-MSMs@PEI-PA-Ti4+ can be used as HILIC or IMAC material to capture N-glycopeptides/phosphopeptides from real biological samples with high efficiency.
using mass spectrometry, Trends Microbiol. 14 (2006) 229–235. [10] B. Domon, R. Aebersold, Review-mass spectrometry and protein analysis, Science 312 (2006) 212–217. [11] X. Xu, C. Deng, M. Gao, W. Yu, P. Yang, X. Zhang, Synthesis of magnetic microspheres with immobilized metal ions for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry, Adv. Mater. 18 (2006) 3289–3293. [12] J. Zhu, Z. Sun, K. Cheng, R. Chen, M. Ye, B. Xu, D. Sun, L. Wang, J. Liu, F. Wang, H. Zou, Comprehensive mapping of protein N-glycosylation in human liver by combining hydrophilic interaction chromatography and hydrazide chemistry, J. Proteome Res. 13 (2014) 1713–1721. [13] C. Chen, W. Su, B. Huang, Y. Chen, H. Tai, R.P. Obena, Interaction modes and approaches to glycopeptide and glycoprotein enrichment, Analyst 139 (2014) 688–704. [14] Z. Wang, N. Lv, W. Bi, J. Zhang, J. Ni, Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis, ACS Appl. Mater. Interfaces 7 (2015) 8377–8392. [15] J. Peng, R. Wu, Metal-organic frameworks in proteomics/peptidomics-a review, Anal. Chim. Acta 1027 (2018) 9–21. [16] H. Zhou, S. Xu, M. Ye, S. Feng, C. Pan, X. Jiang, X. Li, G. Han, Y. Fu, H. Zou, Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis, J. Proteome Res. 5 (2006) 2431–2437. [17] Y. Zhang, C. Chen, H. Qin, R. Wu, H. Zou, The synthesis of Ti-hexagonal mesoporous silica for selective capture of phosphopeptides, Chem. Commun. 46 (2010) 2271–2273. [18] J. Lu, M. Wang, Y. Li, C. Deng, Facile synthesis of TiO2/graphene composites for selective enrichment of phosphopeptides, Nanoscale 4 (2012) 1577–1580. [19] Y. Yan, Z. Zheng, C. Deng, Y. Li, X. Zhang, P. Yang, Hydrophilic polydopaminecoated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis, Anal. Chem. 85 (2013) 8483–8487. [20] Y. Wang, J. Wang, M. Gao, X. Zhang, An ultra hydrophilic dendrimer-modified magnetic graphene with a polydopamine coating for the selective enrichment of glycopeptides, J. Mater. Chem. B 3 (2015) 8711–8716. [21] M. Zhao, C. Deng, X. Zhang, The design and synthesis of a hydrophilic core-shellshell structured magnetic metal-organic framework as a novel immobilized metal ion affinity platform for phosphoproteome research, Chem. Commun. 50 (2014) 6228–6231. [22] Y. Ji, Z. Xiong, G. Huang, J. Liu, Z. Zhang, Z. Liu, J. Ou, M. Ye, H. Zou, Efficient enrichment of glycopeptides using metal-organic frameworks by hydrophilic interaction chromatography, Analyst 139 (2014) 4987–4993. [23] W. Ma, L. Xu, Z. Li, Y. Sun, Y. Bai, H. Liu, Post-synthetic modification of an aminofunctionalized metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Nanoscale 8 (2016) 10908–10912. [24] D. Shen, J. Yang, X. Li, L. Zhou, R. Zhang, W. Li, L. Chen, R. Wang, F. Zhang, D. Zhao, Biphase stratification approach to three-dimensional dendritic biodegradable mesoporous silica nanospheres, Nano Lett. 14 (2014) 923–932. [25] Q. Yue, J. Li, W. Luo, Y. Zhang, A.A. Elzatahry, X. Wang, C. Wang, W. Li, X. Cheng, A. Alghamdi, An interface coassembly in biliquid phase: toward core-shell magnetic mesoporous silica microspheres with tunable pore size, J. Am. Chem. Soc. 137 (2015) 13282–13289. [26] X. Zou, J. Jie, B. Yang, A facile and cheap synthesis of zwitterion coatings of the CS@PGMA@IDA nanomaterial for highly specific enrichment of glycopeptides, Chem. Commun. 52 (2016) 3251–3253. [27] N. Sun, J. Wang, J. Yao, C. Deng, Hydrophilic mesoporous silica materials for highly specific enrichment of N-linked glycopeptide, Anal. Chem. 89 (2017) 1764–1771. [28] W. Ma, Y. Zhang, L. Li, Y. Zhang, M. Yu, J. Guo, H. Lu, C. Wang, Ti4+-immobilized magnetic composite microspheres for highly selective enrichment of phosphopeptides, Adv. Funct. Mater. 23 (2013) 107–115. [29] T. Wu, J. Shi, C. Zhang, L. Zhang, Y. Du, Highly specific phosphopeptide enrichment by titanium(IV) cross-linked chitosan composite, J. Chromatogr. B 1008 (2016) 234–239. [30] X. Zou, J. Jie, B. Yang, Single-step enrichment of N-glycopeptides and phosphopeptides with novel multifunctional Ti4+-immobilized dendritic polyglycerol coated chitosan nanomaterials, Anal. Chem. 89 (2017) 7520–7526. [31] Y. Xie, C. Deng, Designed synthesis of a "One for Two" hydrophilic magnetic aminofunctionalized metal-organic framework for highly efficient enrichment of glycopeptides and phosphopeptides, Sci. Rep. 7 (2017) 1162. [32] D. Xu, G. Yan, M. Gao, C. Deng, X. Zhang, Selective enrichment of glycopeptides/ phosphopeptides using Fe3O4@Au-B(OH)2@mTiO2 core-shell microspheres, Talanta 166 (2017) 154–161. [33] J. Song, B. Zhou, H. Zhou, L. Wu, Q. Meng, Z. Liu, B. Han, Porous zirconium-phytic acid hybrid: a highly efficient catalyst for meerwein-ponndorf-verley reductions, Angew. Chem. Int. Ed. 54 (2015) 9399–9403. [34] L. Li, G. Zhang, Z. Su, One-step assembly of phytic acid metal complexes for superhydrophilic coatings, Angew. Chem. Int. Ed. 55 (2016) 9093–9096. [35] X. Song, Y. Chen, M. Rong, Z. Xie, T. Zhao, Y. Wang, X. Chen, O.S. Wolfbeis, A phytic acid induced super-amphiphilic multifunctional 3D graphene-based foam, Angew. Chem. Int. Ed. 55 (2016) 3936–3941. [36] Y. Hong, C. Pu, H. Zhao, Q. Sheng, Q. Zhan, M. Lan, Yolk-shell magnetic mesoporous TiO2 microspheres with flowerlike NiO nanosheets for highly selective enrichment of phosphopeptides, Nanoscale 9 (2017) 16764–16772. [37] A.K. Meka, P.L. Abbaraju, H. Song, C. Xu, J. Zhang, H. Zhang, M. Yu, C. Yu, A vesicle supra-assembly approach to synthesize amine-functionalized hollow dendritic mesoporous silica nanospheres for protein delivery, Small 12 (2016)
4. Conclusions In summary, magnetic mesoporous silica microspheres with ordered radial mesochannels were successfully synthesized in a chlorobenzenewater mixed system by using TEA as a catalyst, CTAB as a surfactant, TEOS and APTES as silica sources. The obtained Mag-MSMs were further modified with PEI, PA and Ti4+ through LbL assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of immobilized Ti4+, the obtained Mag-MSMs@PEI-PA-Ti4+ could be used as a HILIC material for N-glycopeptides enrichment, as well as an IMAC material for phosphopeptides enrichment. The enrichment results of standard proteins (IgG and α-casein), human serum (2 μL) and HeLa cells extracts (200 μg) demonstrate that the Mag-MSMs@PEI-PA-Ti4+ possess excellent enrichment performance not only for N-glycopeptides but also for phosphopeptides. We believe that this dual-purpose material with outstanding enrichment ability will have great potential in Nglycoproteome and phosphoproteome research. Acknowledgements 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). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2019.01.005. References [1] G. Durand, N. Seta, Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring, Clin. Chem. 46 (2000) 795–805. [2] G. Walsh, R. Jefferis, Post-translational modifications in the context of therapeutic proteins, Nat. Biotechnol. 24 (2006) 1241–1252. [3] J.W. Dennis, I.R. Nabi, M. Demetriou, Metabolism, cell surface organization and disease, Cell 139 (2009) 1229–1241. [4] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [5] J. Roth, Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control and cell interactions, Chem. Rev. 102 (2002) 285–303. [6] J.A. Ludwig, J.N. Weinstein, Biomarkers in cancer staging, prognosis and treatment selection, Nat. Rev. Cancer 5 (2005) 845–856. [7] J. Zhang, S. Pan, Y. Wang, J. Leverenz, E. Peskind, J. Quinn, J. Nutt, J. Jankovic, C. Kenny, J. Jin, J. Li, D. Zhu, C. Pan, C. Zabetian, K. Chung, Identification of glycoproteins in human cerebrospinal fluid as biomarkers for diagnosis and progression of Parkinson's disease, Mol. Cell. Proteom. 5 (2006) 272. [8] T.J. Griffin, D.R. Goodlett, R. Aebersold, Advances in proteome analysis by mass spectrometry, Curr. Opin. Biotechnol. 12 (2001) 607–612. [9] E. Kolker, R. Higdon, J.M. Hogan, Protein identification and expression analysis
84
Talanta 197 (2019) 77–85
Y. Hong et al. 5169–5177. [38] J. Li, F. Wang, H. Wan, J. Liu, Z. Liu, K. Cheng, H. Zou, Magnetic nanoparticles coated with maltose-functionalized polyethyleneimine for highly efficient enrichment of N-glycopeptides, J. Chromatogr. A 1425 (2015) 213–220. [39] X. Sun, J. Dong, J. Li, M. Ye, W. Zhang, J. Ou, Facile preparation of polysaccharide functionalized macroporous adsorption resin for highly selective enrichment of glycopeptides, J. Chromatogr. A 1498 (2017) 72–79. [40] F. Jiao, F. Gao, H. Wang, Y. Deng, Y. Zhang, X. Qian, Y. Zhang, Polymeric hydrophilic ionic liquids used to modify magnetic nanoparticles for the highly selective enrichment of N-linked glycopeptides, Sci. Rep. 7 (2017) 6984. [41] W. Ma, L. Xu, X. Li, S. Shen, M. Wu, Y. Bai, H. Liu, Cysteine-functionalized metalorganic framework: facile synthesis and high efficient enrichment of N-linked glycopeptides in cell lysate, ACS Appl. Mater. Interfaces 9 (2017) 19562–19568. [42] H. Wang, F. Jiao, F. Gao, J. Huang, Y. Zhao, Y. Shen, Y. Zhang, X. Qian, Facile
[43] [44] [45] [46]
85
synthesis of magnetic covalent organic frameworks for the hydrophilic enrichment of N-glycopeptides, J. Mater. Chem. B 5 (2017) 4052–4059. F. Jiao, F. Gao, H. Wang, Y. Deng, Y. Zhang, X. Qian, Y. Zhang, Ultrathin au nanowires assisted magnetic graphene-silica ZIC-HILIC composites for highly specific enrichment of N-linked glycopeptides, Anal. Chim. Acta 970 (2017) 47–56. Y. Li, J. Wang, N. Sun, C. Deng, Glucose-6-phosphate-functionalized magnetic microsphere as novel hydrophilic probe for specific capture of N-linked glycopeptides, Anal. Chem. 89 (2017) 11151–11158. Q. Liu, Y. Xie, C. Deng, Y. Li, One-step synthesis of carboxyl-functionalized metalorganic framework with binary ligands for highly selective enrichment of N-linked glycopeptides, Talanta 175 (2017) 477–482. Y. Hong, Q. Zhan, C. Pu, Q. Sheng, H. Zhao, M. Lan, Highly efficient enrichment of phosphopeptides from HeLa cells using hollow magnetic macro/mesoporous TiO2 nanoparticles, Talanta 187 (2018) 223–230.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Hydrophilic phytic acid-functionalized magnetic dendritic mesoporous silica nanospheres with immobilized Ti4+: A dual-purpose affinity material for highly efficient enrichment of glycopeptides/phosphopeptides Yayun Honga,1, Qiliang Zhana,1, Yu Zhenga, Chenlu Pua, Hongli Zhaoa, , Minbo Lana,b, ⁎
T
⁎⁎
a
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 b State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Magnetic mesoporous silica microspheres Phytic acid LbL assembly N-glycopeptides enrichment Phosphopeptide enrichment
In this work, magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels were fabricated by a self-assembly synthesis in chlorobenzene-water mixed system. Then, the obtained Mag-MSMs were modified with polyethyleneimine (PEI), phytic acid (PA) and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ possessed combined properties of hydrophilic interaction liquid chromatography (HILIC)- and immobilized metal ion affinity chromatography (IMAC)-based materials, which could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The proposed Mag-MSMs@ PEI-PA-Ti4+ exhibited an outstanding performance for N-glycopeptides enrichment (selectivity: IgG/ BSA = 1:1000; sensitivity: 0.5 fmol/μL IgG) and phosphopeptides enrichment (selectivity: α-casein/ BSA=1:5000; sensitivity: 0.2 fmol/μL α-casein). Furthermore, after enrichment with Mag-MSMs@PEI-PA-Ti4+, a total of 276 N-glycopeptides assigned to 132 glycoproteins were identified from 2 μL human serum and 1645 phosphopeptides corresponding to 704 phosphoproteins were identified from 200 μg HeLa cell extracts.
1. Introduction Protein post-translational modifications (PTMs), especially glycosylation and phosphorylation, play a key role in many important biological processes and often participate in the development of various diseases [1–3]. Abnormal glycoprotein and phosphoprotein with high clinical sensitivity and specificity can be used as disease biomarkers for clinical diagnosis [4–7]. Currently, mass spectrometry (MS) is the most powerful platform for protein PTMs research to accurately identify and locate glycosylation/phosphorylation sites [8–10]. However, it is still difficult to study these two PTMs directly with MS due to the severe suppression caused by the coexistence of high abundant unmodified peptides [11,12]. Therefore, an effective enrichment prior to MS analysis is a prerequisite for accurate identification of glycopeptides/ phosphopeptides from biological samples. To date, a lot of affinity materials are available for peptides enrichment, especially nanomaterials have been widely used in
biomolecule enrichment due to their large surface area and easy postfunctionalization [13–15]. Nanomaterials, such as silica [16,17], graphene [18–20] and metal-organic frameworks (MOFs) [21–23] are commonly used as substrates for the synthesis of affinity materials for peptides enrichment. Among them, mesoporous silica has attracted increasing attention in the field of adsorption separation due to its tunable structures and morphologies. However, conventional mesoporous silica with worm-like mesochannels have relatively small pore sizes (< 2.5 nm) [24,25], which will hamper the subsequent modification of the mesopores walls. In this regard, mesoporous silica with ordered radial mesochannels is considered as an alternative. The unique radial mesochannels can provide a large and easily accessible mesopore, which can facilitate the subsequent modification. Therefore, it is of great importance to develop a facile method to synthesize mesoporous silica microspheres with ordered radial mesochannels. Currently, based on the hydrophilicity of glycopeptides, hydrophilic interaction liquid chromatography (HILIC) has matured to enrich N-
Corresponding author. Corresponding author at: 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. E-mail addresses: [email protected] (H. Zhao), [email protected] (M. Lan). 1 Author contributes equally to this work. ⁎
⁎⁎
https://doi.org/10.1016/j.talanta.2019.01.005 Received 29 October 2018; Received in revised form 26 December 2018; Accepted 2 January 2019 Available online 03 January 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 197 (2019) 77–85
Y. Hong et al.
glycopeptides through hydrophilic interaction [26,27]. And based on the reversible affinity between phosphate groups and immobilized metal ions, immobilized metal ion affinity chromatography (IMAC) has been widely applied to phosphopeptide enrichment [28,29]. However, a lot of affinity materials can only be used for separate enrichment of glycopeptides or phosphopeptides. When studying glycopeptides and phosphopeptides simultaneously, two different materials should be prepared, one for glycopeptides enrichment and the other for phosphopeptides enrichment. Thus, dual-purpose materials with combined properties of HILIC and IMAC for N-glycopeptides and phosphopeptides enrichment have been paid much attention [30–32]. Phytic acid (PA), an eco-friendly and renewable compound, is considered as an excellent modification material. On one hand, PA with six phosphate groups can offer strong affinity sites for metal ions binding to form an IMAC affinity material [33–35]. On the other hand, the excellent hydrophilicity of PA can endow affinity material with HILIC performance [34,35]. Therefore, the affinity material modified with PA can be used for both phosphopeptides and glycopeptides enrichment. Herein, we fabricated magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels through a self-assembly synthesis in chlorobenzene-water mixed system, then the Mag-MSMs were modified with polyethyleneimine (PEI), PA and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. The unique radial pore structure facilitated more PA grafting onto the pore walls of mesoporous silica shell, thereby increasing the binding amount of immobilized Ti4+. Due to the excellent hydrophilicity of PEI and PA and a large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The selectivity and sensitivity of the Mag-MSMs@PEIPA-Ti4+ in N-glycopeptides and phosphopeptides enrichment have been evaluated by using human immunoglobulin G (IgG) digest and αcasein digest, respectively. Furthermore, the Mag-MSMs@PEI-PA-Ti4+ also showed a great advantage in capturing N-glycopeptides from human serum and identifying phosphopeptides from HeLa cell extracts, indicating its great potential in protein PTMs research.
Fe3O4@RF in a chlorobenzene-water system. Briefly, 0.1 g Fe3O4@RF was dispersed in 250 mL deionized water by ultrasonication. Then, 0.68 g of TEA was added and the mixture was mechanically stirred at 80 °C for 0.5 h. Afterwards, 1.52 g CTAB were added and kept under stirring for 1 h, followed by the addition of the mixture of 15 mL of chlorobenzene and 100 μL of APTES. The reaction solution was kept under stirring at 80 °C for another 1 h. Finally, 3 mL TEOS were added in the reaction solution and stirred for 24 h. The products were collected with a magnet and washed alternately with ethanol (three times) and deionized water (three times), followed by extraction with ethanol at 65 °C for three times to remove the template and dried in vacuum at 45 °C overnight. 2.3. Synthesis of Mag-MSMs@PEI-PA-Ti4+ PEI, PA and Ti4+ were modified onto the surface of Mag-MSMs via LbL assembly. The dried Mag-MSMs were dispersed in 100 mL PEI aqueous solution (5 mg/mL) by 5 min ultrasonication and stirred for 12 h at room temperature. The obtained nanoparticles were collected by magnetic separation and the excess PEI was removed by three washings with deionized water. After that, Mag-MSMs@PEI were then redispersed in 100 mL PA aqueous solution (8.4 mg/mL) and stirred for 6 h at room temperature, magnetic separation and followed by three washings. Finally, the Mag-MSMs@PEI-PA were incubated in 50 mL Ti (SO4)2 solution (100 mM) for 2 h to immobilize Ti4+. The Mag-MSMs@ PEI-PA-Ti4+ were collected with a magnet and rinsed with deionized water for three times, followed by lyophilization to dryness. 2.4. Characterization of Mag-MSMs@PEI-PA-Ti4+ Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1400 transmission electron microscope (JEOL, Japan). Energy dispersive spectroscopy (EDS) images were conducted on Falion 60S (EDAX, USA). Zeta potential measurements were performed on a Nano-ZS90 instrument (Malvern, UK) in water at 25 °C. The magnetic hysteresis curves were carried out on a Lakeshore 7407 vibrating sample magnetometer (Lakeshore, USA) at room temperature. Nitrogen adsorption-desorption isotherms were measured on an ASAP 2020 apparatus (Micrometritics, USA). Fourier transform infrared (FTIR) spectra were obtained from Nicolet 6700 (Thermo Fisher Scientific, USA) using KBr pellets.
2. Experimental 2.1. Materials and chemicals Triethanolamine (TEA), cetyltrimethylammonium bromide (CTAB), chlorobenzene, 3-aminopropyltriethoxysilane (APTES), tetraethylorthosilicate (TEOS), polyethyleneimine (PEI, 50%, Mw=70000), phytic acid (PA, 70%) and Ti(SO4)2 were purchased from Aladdin (Shanghai, China). Bovine serum albumin (BSA), α-casein (from bovine milk), immunoglobulin G (IgG) from human serum, ammonium bicarbonate (NH4HCO3), urea, dithiothreitol (DTT), iodoacetamide (IAA), trypsin and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptide-N-glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA, USA). Acetonitrile (ACN), trifluoroacetic acid (TFA) and formic acid (FA) were of chromatographic grade. Human serum samples were collected from healthy people and obtained from the Sixth People's Hospital affiliated to Shanghai Jiaotong University according to the standard clinical procedures. Ultrapure water (18.2 MΩ cm) was produced using a Milli-Q system (Millipore, Bedford, MA, USA). All chemicals were used as received without purifcation.
IgG, BSA and α-casein were dissolved in 50 mM NH4HCO3 buffer (pH 8.2) and treated with trypsin (enzyme to protein ratio of 1:25, w/ w) at 37 °C for 17 h for digestion. IgG and BSA were reduced by DTT and alkylated by IAA. α-Casein was digested directly. For preparation of human serum digest, 2 μL of human serum were diluted with 18 μL NH4HCO3 buffer (50 mM, pH 8.2) containing 8 M urea and reduced by DTT at 56 °C for 45 min, and then alkylated by IAA in dark for 30 min. Finally, the solution was diluted to 50 μL with NH4HCO3 buffer and treated with trypsin at 37 °C for 17 h at an enzyme to protein ratio of 1:25 (w/w). Tryptic digest was lyophilized for further use. The proteins from HeLa cells were extracted as previously described [36] and the process of protein digestion was the same as that of IgG.
2.2. Synthesis of Mag-MSMs
2.6. N-glycopeptides and phosphopeptides enrichment
Magnetic Fe3O4 nanoparticles were synthesized by a solvothermal method and then coated with resorcinol-formaldehyde (RF) resin to form magnetic RF resin microspheres (denoted as Fe3O4@RF) through interface sol-gel polymerization of resorcinol and formaldehyde as described by Yue et al. [25]. Subsequently, the mesoporous silica shell with ordered radial mesochannels were coated on the surface of
For glycopeptides enrichment from IgG digest, 20 μg of MagMSMs@PEI-PA-Ti4+ was dispersed in 200 μL of loading buffer (ACN/ H2O/TFA=90/8/2, v/v/v) containing 2 × 10−7 M IgG digest and then incubated at room temperature for 30 min. After that, the microspheres 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,
2.5. Sample preparation
78
Talanta 197 (2019) 77–85
Y. Hong et al.
Scheme 1. Synthetic procedure of Mag-MSMs@PEI-PA-Ti4+.
the captured glycopeptides were eluted with 10 μL 3% TFA aqueous solution for 15 min. The eluent was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For glycopeptides enrichment from human serum digest, 2 μL of the lyophilized serum digest was redissolved in 500 μL of loading buffer (ACN/H2O/TFA = 90/8/2, v/v/v) and then incubated with 400 μg of Mag-MSMs@PEI-PA-Ti4+ for 30 min. Subsequently, the microspheres 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 with 3% TFA aqueous solution (100 μL × 3) and then lyophilized for deglycosylation. The lyophilized glycopeptides were redissolved in 19 μL of 50 mM NH4HCO3 and 1 μL (500 unites) of PNGaseF was added. The mixture was incubated at 37 °C for 16 h and then lyophilized for nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis. For phosphopeptides enrichment from α-casein digest, 20 μg of MagMSMs@PEI-PA-Ti4+ was dispersed in 200 μL of loading buffer (ACN/ H2O/TFA=50/48/2, v/v/v) containing 2 × 10−7 M α-casein digest and then incubated at room temperature for 30 min. After that, the microspheres were collected 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 phosphopeptides enrichment from HeLa cell extracts, 200 μg of tryptic digests were diluted with 500 μL of loading buffer (ACN/H2O/ TFA = 70/29/1, v/v/v) and incubated with 2 mg of Mag-MSMs@PEIPA-Ti4+ for 30 min. Subsequently, the microspheres were collected and rinsed with 500 μL of washing buffer (ACN/H2O/TFA = 70/29.9/0.1, v/v/v) for three times. The captured phosphopeptides were eluted with 0.4 M NH4OH solution (100 μL × 3) and then lyophilized for nano-LCMS/MS analysis directly.
2.7. Mass spectrometry analysis MALDI-TOF MS experiments were carried out in positive ion mode by a 4800 Plus MALDI-TOF MS (AB Sciex, USA) with a Nd:YAG laser emitting at 355 nm. 1 μL analyte was dropped on the plate, then 1 μL matrix (DHB, 25 mg/mL, ACN/H2O/H3PO4 = 70:29:1, v/v/v) was dropped and analyzed by MALDI-TOF MS. Nano-LC-MS/MS experiments were performed using an EASY-nLC 1000 Nano HPLC System (Thermo Fisher Scientific, Bremen, Germany) with a quadrupole-Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Bremen, Germany). The lyophilized 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), then separated using a 60 min gradient. For reversed phase liquid chromatographic (RPLC) separation, 0.1% FA/H2O and 0.1% FA/ACN were used as the mobile phase A and B, respectively. The reversed phase (RP) gradient elution was performed as follows: 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 10 min. The flow rate was maintained at 300 nL/min. All MS and MS/MS spectra were acquired in data-dependent 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 higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28% and the automatic gain control (AGC) target was set to 3 × 105 with a maximum injection time of 45 ms. Dynamic exclusion (30 s) was on. 2.8. Database search and data analysis The MS raw data files were searched by Proteome Discoverer software (Thermo Fisher Scientific, version 1.4) with Sequest HT search 79
Talanta 197 (2019) 77–85
Y. Hong et al.
Ti4+ were confirmed through diverse characterizations. TEM images of the products obtained after each synthesis step are shown in Fig. 1. The water dispersible Fe3O4 nanoparticles synthesized by a solvothermal method possess a uniform size of 250 nm without aggregation (Fig. 1a). After RF resin coating, a RF resin layer with thickness of ~30 nm can be clearly observed on Fe3O4@RF nanoparticles (Fig. 1b), indicating that the RF resin has been successfully modified on Fe3O4 nanoparticles through the interface sol-gel polymerization of resorcinol and formaldehyde. Subsequently, the mesoporous silica shell was formed on Fe3O4@RF nanoparticles in a chlorobenzene-water mixed system. After removal of CTAB through extraction, the Mag-MSMs exhibit distinct core-shell-shell structure with an ordered radial mesochannels (Fig. 1c). With the modification of PEI, PA and Ti4+, the mesoporous channels gradually decreased, indicating that PEI, PA and Ti4+ have been successfully grafted onto the pore walls of mesoporous silica shell via LbL assembly (Fig. 1d–f). The stepwise coating of PEI, PA and Ti4+ on the Mag-MSMs was monitored by observing zeta potential changes of Mag-MSMs after each modification step. As shown in Fig. 2a, the zeta potential of the MagMSMs was −21.9 mV due to the presence of a large number of silicon hydroxyl. After PEI coating, the zeta potential changed from negative to positive, which was attributed to the numerous amino groups presented in PEI. Subsequently, grafting with PA, the zeta potential of the microspheres changed negative again due to the six phosphate groups of PA. Finally, after Ti4+ chelating, the zeta potential of the Mag-MSMs@ PEI-PA-Ti4+ increased to +16.9 mV. The changes of zeta potential indicate the successful introduction of functional group (amino groups, phosphate group and titanium ion) on Mag-MSMs via LbL procedure. In addition, the EDS spectrum (Fig. 2b) and FT-IR spectra (Fig. S1) further confirmed the successful modification on Mag-MSMs. The N2 adsorption-desorption analysis was performed to study the porosity of MagMSMs@PEI-PA-Ti4+ (Fig. 2c). The pore size distribution curve reveals an average pore size of 3.6 nm (Fig. 2c inset), which is suitable for low
engine and the database was the Human UniProtKB/Swiss-Prot database (Release 2018-01-26, with 20189 sequences). Two missed cleavages were allowed by trypsin digestion. The mass tolerances for the precursor ions and fragment ions were set to 10 ppm and 0.05 Da, respectively. False discovery rates (FDR) were set to 1% for both peptide and protein. Carbamidomethyl on cysteine (C) was chosen for fixed modifications, and oxidation on methionine (M) was set as variable modifications. For phosphopeptides, phosphorylation of serine/threonine/tyrosine (S/T/Y) were set as variable modifications. For glycopeptides, deamidation on asparagine (N) was set as variable modifications. Only glycopeptides with the N-glycosylation consensus sequence (N-! Proline-S/T/C) were considered to be reliable. 3. Results and discussion 3.1. Synthesis and characterization of Mag-MSMs@PEI-PA-Ti4+ The synthetic procedure of Mag-MSMs@PEI-PA-Ti4+ is illustrated in Scheme 1. The Mag-MSMs were prepared in a chlorobenzene-water mixed system, using Fe3O4@RF nanoparticles as seeds, TEA as a catalyst, CTAB as a surfactant, TEOS and APTES as silica sources and chlorobenzene as swelling agents. Firstly, a water phase containing Fe3O4@RF nanoparticles, CTAB and TEA was mixed with an oil phase containing APTES and chlorobenzene, then TEOS was added into the mixture. During the reaction process, negatively charged silica oligomers derived from TEOS hydrolysis coassembled with positively charged CTAB micelles and protonated APTES into rod-like composites through electrostatic interaction, and deposited on Fe3O4@RF nanoparticles to form Mag-MSMs [25,37]. The Mag-MSMs were collected with a magnet followed by surfactant extraction in ethanol. Then, PEI, PA and Ti4+ were grafted onto the inwalls of mesoporous channels via LbL assembly at room temperature to obtain Mag-MSMs@PEI-PA-Ti4+. The morphology and surface modification of Mag-MSMs@PEI-PA-
Fig. 1. TEM images of (a) Fe3O4, (b) Fe3O4@RF, (c) Mag-MSMs, (d) Mag-MSMs@PEI, (e) Mag-MSMs@PEI-PA and (f) Mag-MSMs@PEI-PA-Ti4+. 80
Talanta 197 (2019) 77–85
Y. Hong et al.
Fig. 2. (a) Zeta potential changes of microspheres during LbL assembly procedure; (b) EDS spectrum of Mag-MSMs@PEI-PA-Ti4+; (c) N2 adsorption-desorption isotherm and pore size distribution (inset) of Mag-MSMs@PEI-PA-Ti4+; (d) Magnetic hysteresis curves of Fe3O4 and Mag-MSMs@PEI-PA-Ti4+; Inset: magnetic response of Mag-MSMs@PEI-PA-Ti4+ to an external magnet.
molecular weight peptides enrichment. Moreover, the magnetic property of Mag-MSMs@PEI-PA-Ti4+ were also investigated. Fig. 2d shows that the saturation magnetization value of Mag-MSMs@PEI-PA-Ti4+ was measured to be 18.2 emu/g. Although this value was significantly reduced in comparison to that of original Fe3O4 nanoparticles (62.0 emu/g) due to multilayered modification, the Mag-MSMs@PEIPA-Ti4+ could still be quickly separated by using a magnet (as shown in Fig. 2d inset).
by the large amount of non-glycopeptides and high salt content (Fig. 3a). However, after treatment with Mag-MSMs@PEI-PA-Ti4+, a total of thirty-six N-glycopeptides (detailed information see Table S1) were observed with significantly increased signal intensity (Fig. 3b). The number of the N-glycopeptides identified from human IgG digest exceeded those of many previous HILIC nanomaterials [38–40], which is mainly attributed to the excellent hydrophilicity from the numerous amino groups of PEI and phosphate groups of PA. In addition, the selectivity and sensitivity of Mag-MSMs@PEI-PATi4+ for N-glycopeptides enrichment were also investigated. For selectivity evaluation, BSA, an unmodified protein, was digested by trypsin and then doped into IgG digest with different molar ratios to simulate complex samples. As shown in Fig. 4a, direct analysis of IgG and BSA digest mixture with a molar ratio of 1:100, the spectrum was completely occupied by a large amount of high-intensity non-glycopeptides. While after enrichment with Mag-MSMs@PEI-PA-Ti4+, thirtythree N-glycopeptides with high MS intensity were identified in a clean background. Compared with the number of N-glycopeptides identified from standard IgG digest, there is only a slight decrease in the number after 100-fold BSA doping. With the molar ratio of IgG and BSA increased to 1:1000, twenty-two N-glycopeptides with high signal intensity still occupied the spectrum. Compared with previous HILIC materials, such as MIL-101(NH2)@Au-Cys (IgG:BSA = 1:50, identified 11 N-glycopeptides) [41], Fe3O4@TpPa-1 (IgG:BSA = 1:100, identified 8 N-glycopeptides) [42] and GO-Fe3O4/SiO2/AuNWs/L-Cys (IgG:BSA = 1:100, identified 15 N-glycopeptides) [43], Mag-MSMs@PEIPA-Ti4+ indeed exhibit an excellent selectivity for N-glycopeptides enrichment. Furthermore, the detection sensitivity of Mag-MSMs@PEIPA-Ti4+ was also evaluated by different concentrations of IgG digest (2
3.2. Evaluation of the N-glycopeptides/phosphopeptides enrichment performance of Mag-MSMs@PEI-PA-Ti4+ using standard proteins Based on the excellent hydrophilicity endowed by PEI and PA, MagMSMs@PEI-PA-Ti4+ can be used as a HILIC material for N-glycopeptides enrichment through the hydrophilic interaction between the glycan chains of N-glycopeptides and the material. Simultaneously, due to the large amount of Ti4+ chelated by PA, Mag-MSMs@PEI-PA-Ti4+, also as an IMAC material, was anticipated to have excellent performance for phosphopeptides enrichment. Therefore, tryptic digests of standard glycoprotein (human IgG) and phosphoprotein (α-casein) were employed as the corresponding testing samples for N-glycopeptides/phosphopeptides enrichment. A typical procedure with respective enrichment conditions to enrich N-glycopeptides/phosphopeptides from protein tryptic digests is displayed in Scheme 2, including incubation for peptides adsorption, removing nontarget peptides and elution of N-glycopeptides/phosphopeptides. Fig. 3 shows the enrichment results of N-glycopeptides before and after treatment with MagMSMs@PEI-PA-Ti4+. It can be seen that, before enrichment, no Nglycopeptides could be observed due to the severe suppression caused 81
Talanta 197 (2019) 77–85
Y. Hong et al.
Scheme 2. Workflow of target peptides enrichment from protein tryptic digests using Mag-MSMs@PEI-PA-Ti4+.
Fig. 3. MALDI-TOF mass spectra of human IgG digest (a) before and (b) after enrichment by Mag-MSMs@PEI-PA-Ti4+.
Fig. 5. MALDI-TOF mass spectra of human IgG tryptic digest with different concentrations after enrichment with Mag-MSMs@PEI-PA-Ti4+.
fmol/μL, 1 fmol/μL and 0.5 fmol/μL). As shown in Fig. 5a, thirteen Nglycopeptides were easily detected from 2 fmol/μL of IgG digest after enrichment by Mag-MSMs@PEI-PA-Ti4+. Even when the concentration of IgG digest was reduced to 0.5 fmol/μL, six N-glycopeptides could still be clearly observed. The outstanding enrichment selectivity and sensitivity of Mag-MSMs@PEI-PA-Ti4+ are mainly attributed to the excellent hydrophilicity of PEI and PA. These results reveal that the Mag-MSMs@ PEI-PA-Ti4+ can be used as a HILIC material for efficient N-glycopeptides enrichment from complex biological sample, even at low concentration. Since the PA molecule carries with six phosphate groups, it can further serve as chelating ligands for Ti4+ immobilization to form IMAC material. Based on the reversible specific affinity of the Ti4+ to the phosphate groups of phosphopeptides, Mag-MSMs@PEI-PA-Ti4+ are also suitable for phosphopeptides enrichment. The enrichment performance of Mag-MSMs@PEI-PA-Ti4+ for phosphopeptides was investigated by capturing phosphopeptides from α-casein digest. As shown in Fig. 6a, without enrichment, only four phosphopeptides with low signal intensity were observed together with a large number of non-
Fig. 4. MALDI-TOF mass spectra of the tryptic digest mixtures of human IgG and BSA without enrichment at a molar ratio of 1:100 (a), and with the MagMSMs@PEI-PA-Ti4+ enrichment at molar ratios of 1:100 (b) and 1:1000 (c). 82
Talanta 197 (2019) 77–85
Y. Hong et al.
Fig. 6. MALDI-TOF mass spectra of α-casein digest (a) before and (b) after enrichment by Mag-MSMs@PEI-PA-Ti4+. # indicates dephosphorylated peptides.
phosphopeptides. However, after enrichment with Mag-MSMs@PEI-PATi4+, non-phosphopeptides were entirely removed and thirty phosphopeptides (detailed information see Table S2) were identified with greatly improved signals (Fig. 6b), indicating that the Mag-MSMs@PEIPA-Ti4+ could also be used as an IMAC material for phosphopeptides enrichment. The high enrichment efficiency of Mag-MSMs@PEI-PATi4+ toward phosphopeptides is attributed to the large amount of Ti4+ immobilized by PA via strong chelating interaction. To further explore the selectivity of Mag-MSMs@PEI-PA-Ti4+ for phosphopeptides enrichment, α-casein digest doped with different molar ratios of BSA digest was used as samples. For comparison, the direct analysis of α-casein digest mixed with 1000-fold BSA digest is shown in Fig. 7a. Obviously, no phosphopeptides could be observed except a large number of non-phosphopeptides. However, after
Fig. 8. MALDI-TOF mass spectra of α-casein tryptic digest with the concentrations of 1 fmol/μL (a), 0.5 fmol/μL (b) and 0.2 fmol/μL (c) after enrichment with Mag-MSMs@PEI-PA-Ti4+.
enrichment, twenty-four phosphopeptides completely occupied the MS spectrum (Fig. 7b). Even increasing the amount of BSA digest to 5000fold (Fig. 7c), nineteen phosphopeptides could still be clearly observed, indicating an excellent phosphopeptides enrichment selectivity of MagMSMs@PEI-PA-Ti4+. For sensitivity measurement, Mag-MSMs@PEIPA-Ti4+ were applied to capture phosphopeptides from different concentrations of α-casein digest (1 fmol/μL, 0.5 fmol/μL and 0.2 fmol/μL). As shown in Fig. 8a, eight phosphopeptides were identified in 1 fmol/μL α-casein digest. Even when the concentration of α-casein digest was reduced to 0.2 fmol/μL, two phosphopeptides could still be identified with signal to noise ratio over 3 (Fig. 8c). These results reveal that the Mag-MSMs@PEI-PA-Ti4+ can also be used for phosphopeptides enrichment from complex and low-concentration samples. 3.3. Evaluation of the N-glycopeptides/phosphopeptides enrichment efficiency of Mag-MSMs@PEI-PA-Ti4+ using real biological samples As a common clinical specimen, human serum contains abundant glycoprotein biomarkers, which is of great importance for potential disease diagnosis. Effective enrichment and identification of glycopeptides in human serum is a prerequisite for discovering new disease biomarkers. Therefore, peptides mixture of 2 μL healthy human serum treated with trypsin was selected to examine the N-glycopeptides enrichment efficiency of Mag-MSMs@PEI-PA-Ti4+. The enrichment process was profiled in Scheme 2 by using ACN/H2O/TFA (90/8/2, v/v/v) as loading buffer and 3% TFA aqueous solution as eluent. The eluted Nglycopeptide was deglycosylated with PNGase F and then analyzed by nano-LC-MS/MS. The N-glycopeptides with a consensus sequence (N!P-S/T/C) and the deamidation simultaneously occurred on asparagines residues (mass increment, 0.9858 Da) were considered to be reliable. A total of 276 N-glycopeptides derived from 132 glycoproteins were identified from only 2 μL human serum (detailed information see Table S3). The Mag-MSMs@PEI-PA-Ti4+ shows a higher enrichment efficiency than those of previous publications, such as MIL-101(Cr)-maltose (5 μL human serum: 111 glycopeptides, 65 glycoproteins) [23], Fe3O4@G6P microspheres (2 μL human serum: 243 glycopeptides, 92
Fig. 7. MALDI-TOF mass spectra of the tryptic digest mixtures of α-casein and BSA without enrichment at a molar ratio of 1:1000 (a), and with the MagMSMs@PEI-PA-Ti4+ enrichment at molar ratios of 1:1000 (b) and 1:5000 (c). # indicates dephosphorylated peptides. 83
Talanta 197 (2019) 77–85
Y. Hong et al.
glycoproteins) [44] and UiO-66-COOH (2 μL human serum: 255 glycopeptides, 93 glycoproteins) [45]. In addition, for evaluating the enrichment efficiency of MagMSMs@PEI-PA-Ti4+ toward phosphopeptides, HeLa cell extracts were selected as a real biological sample. In this process, 200 μg HeLa cells extracts were enriched by Mag-MSMs@PEI-PA-Ti4+ with ACN/H2O/ TFA (70/29/1, v/v/v) as loading buffer and 0.4 M NH4OH solution as eluent. The obtained phosphopeptides were lyophilized and then analyzed by nano-LC-MS/MS. A total of 1645 phosphopeptides derived from 704 phosphoproteins were finally identified (detailed information see Table S4). Compared with the commercial TiO2 (1266 phosphopeptides derived from 554 phosphoproteins, the result can be found in our previous work [46]), the Mag-MSMs@PEI-PA-Ti4+ has obvious advantages in phosphopeptides enrichment. These enrichment results indicate that the Mag-MSMs@PEI-PA-Ti4+ can be used as HILIC or IMAC material to capture N-glycopeptides/phosphopeptides from real biological samples with high efficiency.
using mass spectrometry, Trends Microbiol. 14 (2006) 229–235. [10] B. Domon, R. Aebersold, Review-mass spectrometry and protein analysis, Science 312 (2006) 212–217. [11] X. Xu, C. Deng, M. Gao, W. Yu, P. Yang, X. Zhang, Synthesis of magnetic microspheres with immobilized metal ions for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry, Adv. Mater. 18 (2006) 3289–3293. [12] J. Zhu, Z. Sun, K. Cheng, R. Chen, M. Ye, B. Xu, D. Sun, L. Wang, J. Liu, F. Wang, H. Zou, Comprehensive mapping of protein N-glycosylation in human liver by combining hydrophilic interaction chromatography and hydrazide chemistry, J. Proteome Res. 13 (2014) 1713–1721. [13] C. Chen, W. Su, B. Huang, Y. Chen, H. Tai, R.P. Obena, Interaction modes and approaches to glycopeptide and glycoprotein enrichment, Analyst 139 (2014) 688–704. [14] Z. Wang, N. Lv, W. Bi, J. Zhang, J. Ni, Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis, ACS Appl. Mater. Interfaces 7 (2015) 8377–8392. [15] J. Peng, R. Wu, Metal-organic frameworks in proteomics/peptidomics-a review, Anal. Chim. Acta 1027 (2018) 9–21. [16] H. Zhou, S. Xu, M. Ye, S. Feng, C. Pan, X. Jiang, X. Li, G. Han, Y. Fu, H. Zou, Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis, J. Proteome Res. 5 (2006) 2431–2437. [17] Y. Zhang, C. Chen, H. Qin, R. Wu, H. Zou, The synthesis of Ti-hexagonal mesoporous silica for selective capture of phosphopeptides, Chem. Commun. 46 (2010) 2271–2273. [18] J. Lu, M. Wang, Y. Li, C. Deng, Facile synthesis of TiO2/graphene composites for selective enrichment of phosphopeptides, Nanoscale 4 (2012) 1577–1580. [19] Y. Yan, Z. Zheng, C. Deng, Y. Li, X. Zhang, P. Yang, Hydrophilic polydopaminecoated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis, Anal. Chem. 85 (2013) 8483–8487. [20] Y. Wang, J. Wang, M. Gao, X. Zhang, An ultra hydrophilic dendrimer-modified magnetic graphene with a polydopamine coating for the selective enrichment of glycopeptides, J. Mater. Chem. B 3 (2015) 8711–8716. [21] M. Zhao, C. Deng, X. Zhang, The design and synthesis of a hydrophilic core-shellshell structured magnetic metal-organic framework as a novel immobilized metal ion affinity platform for phosphoproteome research, Chem. Commun. 50 (2014) 6228–6231. [22] Y. Ji, Z. Xiong, G. Huang, J. Liu, Z. Zhang, Z. Liu, J. Ou, M. Ye, H. Zou, Efficient enrichment of glycopeptides using metal-organic frameworks by hydrophilic interaction chromatography, Analyst 139 (2014) 4987–4993. [23] W. Ma, L. Xu, Z. Li, Y. Sun, Y. Bai, H. Liu, Post-synthetic modification of an aminofunctionalized metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Nanoscale 8 (2016) 10908–10912. [24] D. Shen, J. Yang, X. Li, L. Zhou, R. Zhang, W. Li, L. Chen, R. Wang, F. Zhang, D. Zhao, Biphase stratification approach to three-dimensional dendritic biodegradable mesoporous silica nanospheres, Nano Lett. 14 (2014) 923–932. [25] Q. Yue, J. Li, W. Luo, Y. Zhang, A.A. Elzatahry, X. Wang, C. Wang, W. Li, X. Cheng, A. Alghamdi, An interface coassembly in biliquid phase: toward core-shell magnetic mesoporous silica microspheres with tunable pore size, J. Am. Chem. Soc. 137 (2015) 13282–13289. [26] X. Zou, J. Jie, B. Yang, A facile and cheap synthesis of zwitterion coatings of the CS@PGMA@IDA nanomaterial for highly specific enrichment of glycopeptides, Chem. Commun. 52 (2016) 3251–3253. [27] N. Sun, J. Wang, J. Yao, C. Deng, Hydrophilic mesoporous silica materials for highly specific enrichment of N-linked glycopeptide, Anal. Chem. 89 (2017) 1764–1771. [28] W. Ma, Y. Zhang, L. Li, Y. Zhang, M. Yu, J. Guo, H. Lu, C. Wang, Ti4+-immobilized magnetic composite microspheres for highly selective enrichment of phosphopeptides, Adv. Funct. Mater. 23 (2013) 107–115. [29] T. Wu, J. Shi, C. Zhang, L. Zhang, Y. Du, Highly specific phosphopeptide enrichment by titanium(IV) cross-linked chitosan composite, J. Chromatogr. B 1008 (2016) 234–239. [30] X. Zou, J. Jie, B. Yang, Single-step enrichment of N-glycopeptides and phosphopeptides with novel multifunctional Ti4+-immobilized dendritic polyglycerol coated chitosan nanomaterials, Anal. Chem. 89 (2017) 7520–7526. [31] Y. Xie, C. Deng, Designed synthesis of a "One for Two" hydrophilic magnetic aminofunctionalized metal-organic framework for highly efficient enrichment of glycopeptides and phosphopeptides, Sci. Rep. 7 (2017) 1162. [32] D. Xu, G. Yan, M. Gao, C. Deng, X. Zhang, Selective enrichment of glycopeptides/ phosphopeptides using Fe3O4@Au-B(OH)2@mTiO2 core-shell microspheres, Talanta 166 (2017) 154–161. [33] J. Song, B. Zhou, H. Zhou, L. Wu, Q. Meng, Z. Liu, B. Han, Porous zirconium-phytic acid hybrid: a highly efficient catalyst for meerwein-ponndorf-verley reductions, Angew. Chem. Int. Ed. 54 (2015) 9399–9403. [34] L. Li, G. Zhang, Z. Su, One-step assembly of phytic acid metal complexes for superhydrophilic coatings, Angew. Chem. Int. Ed. 55 (2016) 9093–9096. [35] X. Song, Y. Chen, M. Rong, Z. Xie, T. Zhao, Y. Wang, X. Chen, O.S. Wolfbeis, A phytic acid induced super-amphiphilic multifunctional 3D graphene-based foam, Angew. Chem. Int. Ed. 55 (2016) 3936–3941. [36] Y. Hong, C. Pu, H. Zhao, Q. Sheng, Q. Zhan, M. Lan, Yolk-shell magnetic mesoporous TiO2 microspheres with flowerlike NiO nanosheets for highly selective enrichment of phosphopeptides, Nanoscale 9 (2017) 16764–16772. [37] A.K. Meka, P.L. Abbaraju, H. Song, C. Xu, J. Zhang, H. Zhang, M. Yu, C. Yu, A vesicle supra-assembly approach to synthesize amine-functionalized hollow dendritic mesoporous silica nanospheres for protein delivery, Small 12 (2016)
4. Conclusions In summary, magnetic mesoporous silica microspheres with ordered radial mesochannels were successfully synthesized in a chlorobenzenewater mixed system by using TEA as a catalyst, CTAB as a surfactant, TEOS and APTES as silica sources. The obtained Mag-MSMs were further modified with PEI, PA and Ti4+ through LbL assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of immobilized Ti4+, the obtained Mag-MSMs@PEI-PA-Ti4+ could be used as a HILIC material for N-glycopeptides enrichment, as well as an IMAC material for phosphopeptides enrichment. The enrichment results of standard proteins (IgG and α-casein), human serum (2 μL) and HeLa cells extracts (200 μg) demonstrate that the Mag-MSMs@PEI-PA-Ti4+ possess excellent enrichment performance not only for N-glycopeptides but also for phosphopeptides. We believe that this dual-purpose material with outstanding enrichment ability will have great potential in Nglycoproteome and phosphoproteome research. Acknowledgements 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). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2019.01.005. References [1] G. Durand, N. Seta, Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring, Clin. Chem. 46 (2000) 795–805. [2] G. Walsh, R. Jefferis, Post-translational modifications in the context of therapeutic proteins, Nat. Biotechnol. 24 (2006) 1241–1252. [3] J.W. Dennis, I.R. Nabi, M. Demetriou, Metabolism, cell surface organization and disease, Cell 139 (2009) 1229–1241. [4] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [5] J. Roth, Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control and cell interactions, Chem. Rev. 102 (2002) 285–303. [6] J.A. Ludwig, J.N. Weinstein, Biomarkers in cancer staging, prognosis and treatment selection, Nat. Rev. Cancer 5 (2005) 845–856. [7] J. Zhang, S. Pan, Y. Wang, J. Leverenz, E. Peskind, J. Quinn, J. Nutt, J. Jankovic, C. Kenny, J. Jin, J. Li, D. Zhu, C. Pan, C. Zabetian, K. Chung, Identification of glycoproteins in human cerebrospinal fluid as biomarkers for diagnosis and progression of Parkinson's disease, Mol. Cell. Proteom. 5 (2006) 272. [8] T.J. Griffin, D.R. Goodlett, R. Aebersold, Advances in proteome analysis by mass spectrometry, Curr. Opin. Biotechnol. 12 (2001) 607–612. [9] E. Kolker, R. Higdon, J.M. Hogan, Protein identification and expression analysis
84
Talanta 197 (2019) 77–85
Y. Hong et al. 5169–5177. [38] J. Li, F. Wang, H. Wan, J. Liu, Z. Liu, K. Cheng, H. Zou, Magnetic nanoparticles coated with maltose-functionalized polyethyleneimine for highly efficient enrichment of N-glycopeptides, J. Chromatogr. A 1425 (2015) 213–220. [39] X. Sun, J. Dong, J. Li, M. Ye, W. Zhang, J. Ou, Facile preparation of polysaccharide functionalized macroporous adsorption resin for highly selective enrichment of glycopeptides, J. Chromatogr. A 1498 (2017) 72–79. [40] F. Jiao, F. Gao, H. Wang, Y. Deng, Y. Zhang, X. Qian, Y. Zhang, Polymeric hydrophilic ionic liquids used to modify magnetic nanoparticles for the highly selective enrichment of N-linked glycopeptides, Sci. Rep. 7 (2017) 6984. [41] W. Ma, L. Xu, X. Li, S. Shen, M. Wu, Y. Bai, H. Liu, Cysteine-functionalized metalorganic framework: facile synthesis and high efficient enrichment of N-linked glycopeptides in cell lysate, ACS Appl. Mater. Interfaces 9 (2017) 19562–19568. [42] H. Wang, F. Jiao, F. Gao, J. Huang, Y. Zhao, Y. Shen, Y. Zhang, X. Qian, Facile
[43] [44] [45] [46]
85
synthesis of magnetic covalent organic frameworks for the hydrophilic enrichment of N-glycopeptides, J. Mater. Chem. B 5 (2017) 4052–4059. F. Jiao, F. Gao, H. Wang, Y. Deng, Y. Zhang, X. Qian, Y. Zhang, Ultrathin au nanowires assisted magnetic graphene-silica ZIC-HILIC composites for highly specific enrichment of N-linked glycopeptides, Anal. Chim. Acta 970 (2017) 47–56. Y. Li, J. Wang, N. Sun, C. Deng, Glucose-6-phosphate-functionalized magnetic microsphere as novel hydrophilic probe for specific capture of N-linked glycopeptides, Anal. Chem. 89 (2017) 11151–11158. Q. Liu, Y. Xie, C. Deng, Y. Li, One-step synthesis of carboxyl-functionalized metalorganic framework with binary ligands for highly selective enrichment of N-linked glycopeptides, Talanta 175 (2017) 477–482. Y. Hong, Q. Zhan, C. Pu, Q. Sheng, H. Zhao, M. Lan, Highly efficient enrichment of phosphopeptides from HeLa cells using hollow magnetic macro/mesoporous TiO2 nanoparticles, Talanta 187 (2018) 223–230.