G Model

ARTICLE IN PRESS

CHROMA-357281; No. of Pages 8

Journal of Chromatography A, xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides Gang-Tian Zhu a,b , Xiao-Mei He a , Xi Chen c , Dilshad Hussain a,d , Jun Ding a , Yu-Qi Feng a,∗ a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China b Key Laboratory of Tectonics and Petroleum Resources (Ministry of Education), China University of Geosciences, Wuhan 430075, PR China c Wuhan Institute of Biotechnology, Wuhan 430072, PR China d Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan

a r t i c l e

i n f o

Article history: Received 30 November 2015 Received in revised form 26 January 2016 Accepted 30 January 2016 Available online xxx Keywords: Graphitic carbon nitride Magnetic Phosphopeptide Anion-exchange chromatography pH

a b s t r a c t Anion-exchange chromatography (AEX) is one of the chromatography-based methods effectively being used for phosphopeptide enrichment. However, the development of AEX materials with high specificity toward phosphopeptides is still less explored as compared to immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). In this work, magnetic graphitic carbon nitride (MCN) was successfully prepared and introduced as a promising AEX candidate for phosphopeptide enrichment. Due to the extremely abundant content of nitrogen with basic functionality on the surface, this material kept excellent retention for phosphopeptides at pH as low as 1.8. Benefiting from the large binding capacity at such low pH, MCN showed remarkable specificity to capture phosphopeptides from tryptic digests of standard protein mixtures as well as nonfat milk and human serum. In addition, MCN was also applied to selective enrichment of phosphopeptides from the tryptic digests of rat brain lysate and 2576 unique phosphopeptides were successfully identified. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Phosphorylation is one of the most prominent post-translational modifications of proteins, which is involved in many biological processes including cell proliferation, differentiation, development and apoptosis [1–5]. Mass spectrometry (MS), including matrix-assisted laser desorption/ionization (MALDI) MS or electrospray ionization (ESI) MS, has been a central tool in shotgun based phosphoproteome analysis [6–9]. Phosphopeptide enrichment is a critical step to effectively decrease the complexity of proteome samples prior to MS analysis [10,11]. Until now, many approaches have been developed to enrich phosphopeptides, such as antibody-based method [12,13], chemical derivatization method [14,15] and affinity chromatography-based method [16,17]. The chromatography-based method mainly includes immobilized metal affinity chromatography (IMAC) [18–20], metal oxide affinity chromatography (MOAC) [21–23], cation-exchange chromatography [24] and anion-exchange chromatography (AEX) [25,26]. In most cases, single method cannot achieve desired goals

∗ Corresponding author. Fax: +86 27 68755595. E-mail address: [email protected] (Y.-Q. Feng).

due to the complexity of proteome sample and the wide range of physical and chemical properties of different phosphopeptides. In addition, each affinity sorbent has its own bias on subset of phosphopeptides [27]. It means that combination of multiple methods can effectively increase the phosphoproteome coverage and development of new types of sorbents may produce complementary or even better results to those of the existing methods. Since phosphopeptides contain extra negative charge, AEX has been previously used to fractionate or enrich phosphopeptides because of the stronger retention of phosphopeptides compared to non-phosphopeptides [28,29]. Several reported AEX materials are also available as commercial AEX columns [30–32]. To improve the specificity, IMAC or MOAC is often needed to further purify the fractions eluted from AEX column [33,34]. Dong et al. prepared an organic-silica hybrid AEX monolithic capillary to enrich phosphopeptides from protein digests [28] where pH of loading solution was kept around 8 and the elution was carried out by 5% formic acid. Similarly, Atakay et al. investigated amine-functionalized silica gel for the enrichment of phosphopeptides [35]. The pH of loading solution and eluting solution was set at 4 and 1, respectively. Both of the two AEX materials showed unsatisfactory specificity for phosphopeptides.

http://dx.doi.org/10.1016/j.chroma.2016.01.080 0021-9673/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

2

The pH of solution is the key factor in specific enrichment of phosphopeptides by AEX. At a pH greater than 5, phosphopeptides are well-retained on AEX sorbent, while acidic non-phosphopeptides can be also adsorbed onto AEX. At a pH lower than 2, carboxyl groups are mostly uncharged and acidic non-phosphopeptides are hardly adsorbed on AEX. However, phosphate residues have just a single negative charge at such low pH, so the retention of phosphopeptides on AEX would weaken [29]. Therefore, development of AEX material with high specificity for phosphopeptides is still a challenge and it is desirable to introduce new AEX materials that can keep sustainable retention for phosphopeptides at low pH. Graphitic carbon nitride (g-C3 N4 ) is an analog of graphite consisting of C, N and some impurity H [36–38]. It is widely accepted that g-C3 N4 is composed of tri-s-triazine rings crosslinked by trigonal nitrogen atoms which has unique electronic property and basic surface [39,40]. In addition, g-C3 N4 possesses high thermal stability and is insoluble in water or organic solvent [41]. As a result, g-C3 N4 has been developed as a hot material in many applications especially catalysis [42]. Indeed, it has been used as a solid base catalyst for a variety of reactions [41,43,44]. Besides, Lin et al. utilized g-C3 N4 as a matrix for negative ion MALDI-MS, suggesting that g-C3 N4 can act as a solid base and promote the charging process in the negative ion mode [45]. These previous reports inspired us to explore g-C3 N4 as a potential adsorbent for selective capture of phosphopeptides. Here, for the first time, g-C3 N4 was developed as an AEX material for efficient enrichment of phosphopeptides. The mechanism of phosphopeptide enrichment with g-C3 N4 was investigated and compared with amine-functionalized silica. Additionally, to simplify and expedite the enrichment procedure, we endowed the g-C3 N4 sorbent with magnetic property. In previous work, magnetic g-C3 N4 material, e.g., g-C3 N4 –Fe3 O4 , was prepared by physical blending of Fe3 O4 and g-C3 N4 [46] or chemical deposition of Fe3 O4 nanoparticles onto g-C3 N4 [47–49]. Whereas, the bare Fe3 O4 on the surface of g-C3 N4 makes it unstable in acidic solution, and can lead to nonspecific adsorption. In this article, we designed a facile method to prepare magnetic g-C3 N4 by in-situ thermal polycondensation, in which g-C3 N4 was immobilized on Fe3 O4 @SiO2 particles. The magnetic g-C3 N4 sorbent (MCN) was stable in acidic solution and was able to minimize the nonspecific adsorption. To investigate the performance of the resulting MCN, it was applied to enrich phosphopeptides from tryptic digests of standard protein mixtures, nonfat milk and human serum. Furthermore, MCN was applied to selective enrichment of phosphopeptides from tryptic digests of rat brain lysate.

2. Experimental 2.1. Chemicals and materials Melamine, ferric trichloride hexahydrate (FeCl3 ·6H2 O), sodium acetate (NaAc), ethylene glycol (EG), ethylene diamine, ethanol (EtOH) and ammonia hydrate (NH3 ·H2 O, 25 wt.% in H2 O) were supplied by Shanghai General Chemical Reagent Factory (Shanghai, China). Tetraethyl orthosilicate (TEOS) was obtained from Chemical Plant of Wuhan University (Wuhan, China). HPLC grade acetonitrile (ACN) was obtained from Fisher Scientific (Pittsburgh, USA). Amine-functionalized silica was purchased from Weltech (Wuhan, China). Commercial TiO2 (T104936) was purchased from Aladdin Chemical Reagent (Shanghai, China). Trifluoroacetic acid (TFA), 2,5-dihydroxybenzoic acid (2,5-DHB), bovine ␤-casein and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (St. Louis, USA). Sequencing grade trypsin was obtained from Promega (Madison, WI, USA). Purified water was obtained with a

Milli-Q apparatus (Millipore, Bedford, MA, USA). Nonfat milk was purchased from a local supermarket. Human serum sample was obtained from Wuhan Zhongnan Hospital according to their standard clinical procedures and stored at −70 ◦ C until use. 2.2. Preparation of Fe3 O4 @SiO2 Magnetic core–shell material (Fe3 O4 @SiO2 ) was synthesized by a two step process, according to our previous works. Initially, Fe3 O4 microspheres were prepared by solvothermal reaction [50] and silica shells were coated onto Fe3 O4 microspheres through Stöber method with some modifications [51]. 2.3. Preparation of pure g-C3 N4 and MCN Melamine was chosen as the precursor for synthesis of g-C3 N4 . Pure g-C3 N4 was prepared by direct heating of melamine at 550 ◦ C for 4 h under inert atmosphere [52]. MCN was synthesized by an insitu chemical vapor deposition method. Typically, 1 g of melamine and 0.5 g of Fe3 O4 @SiO2 were added to a mortar and grounded into homogeneous fine powder, and the resulting powder was transferred into a crucible and heated at 550 ◦ C for 4 h under inert atmosphere to obtain MCN. Notably, the crucible was filled up to obtain homogeneous deposition. 2.4. Characterization of the prepared materials Transmission electron microscopy (TEM) images were obtained from JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan). Thermo-gravimetric analysis (TGA) was performed on NETZSCH STA449C thermal analyzer (Bavaria, Germany) under air flowing. The powder X-ray diffraction (XRD) measurements were recorded on a D/MAX-RB X-ray powder diffractometer (RIGAKU, Tokyo, Japan) using Cu K␣ radiation ( = 1.5406 Å) with scattering angles (2) of 1–6◦ . Fourier transform infrared spectrum (FT-IR) was performed with a Thermo Nicolet 670 FT-IR instrument (Boston, MA, USA). 2.5. Preparation of samples Bovine ␤-casein was originally prepared into stock solutions of 1 mg/mL. Proteins were digested with trypsin using an enzyme to substrate ratio of 1:50 (w/w) in 100 mM Tris–HCl (pH 8.5) and the digestion was performed at 37 ◦ C for 16 h. Similarly, BSA (1 mg) was dissolved in 100 ␮L of denaturing buffer solution (8 M urea in 100 mM Tris–HCl, pH 8.5). The protein solution was mixed with 5 ␮L of 100 mM tri(2chloroethyl)phosphate (TCEP) and incubated for 20 min at room temperature to reduce protein disulfide bonding. Iodoacetamide (IAA) (3 ␮L of 500 mM stock) was added to the solution and incubated for an additional 30 min at 25 ◦ C in dark. The reduced and alkylated protein mixture was diluted with 300 ␮L of 100 mM Tris–HCl (pH 8.5). Then, 9 ␮L of 100 mM CaCl2 was added to the above solution and the mixture was digested with trypsin at an enzyme to substrate ratio of 1:50 (w/w) by incubating at 37 ◦ C for 16 h. All the tryptic digests were lyophilized to dryness and stored at −80 ◦ C for further use. For in-solution digestion, nonfat milk (50 ␮L) was first denatured by the ammonium bicarbonate solution (50 mM, 250 ␮L) containing urea (8 M) and incubated at 37 ◦ C for 30 min. Then, dithiothreitol (DTT) solution (200 mM, 25 ␮L) was added and incubated at 55 ◦ C for 1 h. After cooling to room temperature, the IAA solution (200 mM, 50 ␮L) was added and the mixture was kept in the dark for 3 h. Finally, the resulting mixture was incubated with trypsin (2 mg/mL, 5 ␮L) at 37 ◦ C for 16 h. The tryptic digests were stored at −80 ◦ C until further use.

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

Sprague Dawley (SD) male rat were purchased from Hubei Provincial Centre for Disease Control and Prevention (Wuhan, China) and kept separately under a temperature (23 ◦ C) and lighting-controlled house (12 h of light and dark) with free access to a standard chow and water. At 3 or 4 months of age, about 250–300 g of weight, rat was sacrificed and the brain was quickly excised. After perfusing with ice-cold physiological saline to remove blood, the brain tissues were minced with scissors and homogenized in a Potter–Elvejhem homogenizer with a teflon piston on ice for 30 min. 4 mL of RIPA lysis buffer (1% NP-40, 0.25% deoxycholate) with protease and phosphatase inhibitors was used for 0.5 g of tissue lysis. The suspension was homogenized for 20 times and vortexed on ice for 30 min followed by centrifugation at 15,000 × g under 4 ◦ C for 30 min. The protein concentration of the supernatant was measured using Coomassie light blue staining. Proteins were precipitated with some precipitating agents (50% acetone, 49.9% ethanol, 0.1% acetic acid, v/v/v) on ice for 3 h and then centrifuged at 4000 × g under 4 ◦ C for 30 min. The protein pellet was re-suspended in Tris–HCl buffer (8 M urea, 0.2 M Tris, 4 mM CaCl2 , pH 8.0) to make the final concentration of protein at 5 mg/mL. Further digestion procedure was the same as that of BSA and the digested products were desalted by C18 cartridge and stored at −80 ◦ C until use. 2.6. Phosphopeptide enrichment with g-C3 N4 Initially, pure g-C3 N4 was used to deal with synthetic phosphopeptides and tryptic digests of ␤-casein, to optimize the enrichment conditions. Different loading buffers were investigated to achieve more specific enrichment of phosphopeptides. Typically, 1 mg of gC3 N4 was dispersed in 50 ␮L of protein digests in loading buffer and incubated at 25 ◦ C for 5 min, the supernatant was removed after centrifugation at 12,000 × g for 3 min. After washing twice with loading buffer, the trapped peptides were eluted with 50 ␮L of eluting buffer (50% ACN, 45% H2 O, 5% NH3 ·H2 O, v/v/v). The eluted solution was lyophilized to dryness and redissolved in 5 ␮L of matrix solution (20 mg/mL 2,5-DHB in 50% ACN, 49% H2 O, 1% phosphoric acid, v/v/v) and 1 ␮L of the mixture was used for MALDI-MS analysis. Same enrichment protocol was used for amine-functionalized silica. 2.7. Phosphopeptide enrichment with MCN Briefly, 5 ␮L of MCN suspension (60 mg/mL) was added to 50 ␮L of peptide mixtures in loading buffer (50% ACN, 49.9% H2 O, 0.1% TFA, v/v/v) and vortexed at 25 ◦ C for 3 min. After washing twice with 50 ␮L of loading buffer, the trapped phosphopeptides were eluted with 50 ␮L of eluting buffer (50% ACN, 45% H2 O, 5% NH3 ·H2 O, v/v/v). During the procedure, magnetic materials and the captured phosphopeptides were separated from the sample solution by applying an external magnet. The eluted solution was lyophilized to dryness and redissolved by 5 ␮L of matrix solution and 1 ␮L of the mixtures was used for MALDI-MS analysis. Similarly, for phosphopeptide enrichment from tryptic digests of nonfat milk, 1 ␮L of milk digests was diluted 1000 folds with loading buffer and 50 ␮L of the diluted solution was used for phosphopeptide enrichment by MCN. For endogenous phosphopeptide capture from human serum, 2 ␮L of original serum was diluted to 50 ␮L with loading buffer as sampling solution. For phosphopeptide enrichment from tryptic digests of rat brain lysate, 0.5 mg of tryptic digests were diluted to 50 ␮L with loading buffer (50% ACN, 49.9% H2 O, 0.1% TFA, v/v/v). The following sample processing steps were the same as the above procedure. The eluted solution was then lyophilized to dryness, desalted with Zip-Tip C18 and used for RPLC-ESI-MS/MS analysis.

3

2.8. Phosphopeptide enrichment with commercial TiO2 For comparison, commercial TiO2 powder was used to enrich phosphopeptides from tryptic digests of rat brain lysate. Briefly, 1 mg of TiO2 powder was dispersed in 50 ␮L of loading buffer (50% ACN, 49% H2 O, 1% TFA, v/v/v) containing 0.5 mg of tryptic digests. Mixture was incubated at 25 ◦ C for 15 min and the supernatant was removed after centrifugation at 12,000 × g for 3 min. After washing twice with loading buffer, the trapped peptides were eluted with 50 ␮L of 5% aqueous ammonia solution. The eluted solution was then lyophilized to dryness, desalted with Zip-Tip C18 and used for RPLC-ESI-MS/MS analysis. 2.9. Mass spectrometry analysis All MALDI-TOF-MS spectra were recorded with a Shimadzu Axima TOF2 mass spectrometry (Kyoto, Japan). The instrument was equipped with a 337 nm nitrogen laser with a 3 ns pulse width. The detection was performed in positive ion reflector mode with an accelerating voltage of 20 kV and 200 laser shots were averaged to generate each spectrum. RPLC-ESI-MS/MS was used to analyze the sample from rat brain. The analysis was carried out on a hybrid quadrupole-TOF LC–MS/MS mass spectrometer (TripleTOF 5600+, AB Sciex, Massachusetts, USA) equipped with a nanospray source. Peptides were first loaded onto a C18 trap column (5 mm × 0.3 mm i.d., 5 ␮m, Agilent Technologies) and then eluted into a C18 analytical column (150 mm × 75 ␮m i.d., 3 ␮m, 100 Å, Eksigent). Mobile phase A (3% DMSO, 96.9% H2 O, 0.1% formic acid, v/v/v) and mobile phase B (3% DMSO, 96.9% ACN, 0.1% formic acid, v/v/v) were used to establish a 100 min gradient, which was comprised of 0–65 min of 5–23% B, 65–85 min of 23–52% B, 85–86 min of 52–80% B, 86–90 min of 80% B, 90–90.1 min of 80–5% B, and 90.1–100 min of 5% B. A constant flow rate was set at 300 nL/min. MS scans were conducted from 350 to 1500 amu, with a 250 ms time span. For MS/MS analysis, each scan cycle consisted of one full-scan mass spectrum (with m/z ranging 350–1500 and charge states from 2 to 5) followed by 40 MS/MS events. The threshold count was set to 120 to activate MS/MS accumulation and former target ion exclusion was set for 18 s. Raw data from TripleTOF 5600+ were analyzed with ProteinPilot Software 4.5. Data were searched against the Uniprot rat reference proteome database (version 201412) using the following parameters: sample type, identification; cys alkylation, iodoacetamide; digestion, trypsin; special factors, phosphoryl-ation emphasis. Search effort was set to rapid ID. A 1% Critical False Discovery Rates in Protein Pilot was selected to calculate the number of identifications. 3. Results and discussion 3.1. Preparation and characterization of MCN The synthesis procedure of g-C3 N4 and MCN is shown in Fig. 1. Formation of g-C3 N4 was based on thermal polycondensation of melamine. At the reaction temperature as high as 550 ◦ C, the monomers were in the gas phase, allowing convenient deposition of g-C3 N4 on the matrix [38,43], namely Fe3 O4 @SiO2 microspheres, to obtain MCN. Such a chemical vapor deposition method [53] could ensure the sufficient coating of g-C3 N4 onto Fe3 O4 @SiO2 , which can minimize the nonspecific adsorption when MCN was used as a sorbent. The morphology of the fabricated materials was investigated by TEM. According to Fig. 2a, the core/shell structure of Fe3 O4 @SiO2 spheres can be clearly distinguished. The average diameter of

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8 4

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

Fig. 1. The procedure for preparation of g-C3 N4 and MCN.

magnetic core is about 180 nm and the thickness of the silica shell is around 30 nm. Pure g-C3 N4 showed amorphous morphology consisting of transparent sheets (Fig. 2b). From the TEM image of MCN (Fig. 2c), it can be seen that magnetic particles were encapsulated by g-C3 N4 sheets. This meant that Fe3 O4 was coated twice by SiO2 followed by g-C3 N4 , which made MCN stable in acidic solution and able to minimize the nonspecific adsorption caused by bare Fe3 O4 . The composition of the materials was examined by XRD (Fig. S1). The XRD pattern of g-C3 N4 contained a characteristic (0 0 2) interlayer-stacking peak [54]. For Fe3 O4 @SiO2 , strong peaks corresponding to Fe3 O4 were observed. The XRD pattern of MCN contained the characteristic peaks reflected from g-C3 N4 and Fe3 O4 @SiO2 , and the peaks were weaker, demonstrating that MCN was a two-phase composite composed of g-C3 N4 and Fe3 O4 @SiO2 . Furthermore, FT-IR spectra (Fig. S2) also confirmed the successful deposition of g-C3 N4 onto Fe3 O4 @SiO2 . The spectrum of MCN showed the strong absorption band in the range of 1200–1700 cm−1 assigned to the stretching mode of C N heterocycles and the sharp peak centered at 808 cm−1 corresponding to the characteristic breathing mode of the triazine unit [48,55]. Fig. S3 shows the TGA curves of Fe3 O4 @SiO2 and MCN. Approximately 4.6% and 59.0% mass loss were observed for Fe3 O4 @SiO2 and MCN, respectively, which indicated the high content of g-C3 N4 in MCN. 3.2. Mechanism of phosphopeptide enrichment with g-C3 N4 To investigate the mechanism and optimize the conditions of phosphopeptide enrichment, pure g-C3 N4 was first used to extract phosphopeptides from tryptic digests of ␤-casein in different

loading buffers. The detailed conditions and results are shown in Fig. 3. Initially, when water was applied as the loading buffer, both phosphopeptides as well as non-phosphopeptides were extracted by g-C3 N4 ; while only phosphopeptides were trapped on g-C3 N4 when the loading buffer composition was changed to 50% ACN. It can be assumed that the existence of hydrophobic along with the ion-exchange interactions between g-C3 N4 and peptides in water contributed much toward the retention of non-phosphopeptides. But ion-exchange interaction would predominate in the loading buffer containing 50% ACN and therefore, only acidic peptides retained well on g-C3 N4 under these conditions. Changing the TFA content in loading solution brought some significant changes in the mass spectrum (the number and signal intensities of detected phosphopeptides). When the loading buffer was ACN/H2 O (1/1, v/v), ACN/0.2% TFA (1/1, v/v) or ACN/1% TFA (1/1, v/v), five phosphopeptides were observed in the mass spectrum. Further increase in TFA concentration led to loss of some phosphopeptide signals. But even when the loading buffer was ACN/4% TFA (1/1, v/v), one phosphopeptide with high resolution could still be detected, indicating the strong interaction between phosphopeptide and g-C3 N4 . To further study the effect of the TFA content in loading buffer, a synthetic phosphopeptide (SPVLAEDpSEGEG, P1) was enriched with g-C3 N4 and another synthetic phosphopeptide (SPVLADDpSEGEG, P2) as the internal standard during the MALDI-MS analysis. The sequence of P1 is similar to that of P2 with one residue difference; P1 has a methylene group more than P2. The same concentration of P1 and P2 showed almost identical MS responses (Fig. S4), corresponding to the previous works [56], providing a rational foundation for MS quantification with non-isotopic labeling

Fig. 2. TEM images of Fe3 O4 @SiO2 (a), g-C3 N4 (b) and MCN (c).

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

5

Fig. 3. MALDI mass spectra of tryptic digests of ␤-casein after enrichment by g-C3 N4 in different loading buffers including H2 O (a), ACN/H2 O (1/1, v/v) (b), ACN/0.2% TFA (1/1, v/v) (c), ACN/1% TFA (1/1, v/v) (d), ACN/2% TFA (1/1, v/v) (e), ACN/4% TFA (1/1, v/v) (f). The concentration of tryptic digests of ␤-casein was 1.0 × 10−7 M. The phosphopeptides are marked with “ ␤n”.

internal standard. P1 was enriched by g-C3 N4 in five different loading buffers and each experiment was repeated three times. As shown in Fig. S5a, the average recovery (95%) of phosphopeptide in ACN/0.2% TFA (1/1, v/v) was approximate to that (97%) in ACN/H2 O (1/1, v/v). When the TFA content continued to increase, the percent recovery decreased. Considering the recovery and the specificity in complex samples, ACN/0.2% TFA (1/1, v/v) was selected as the loading buffer in further experiments. The pH of this optimized loading buffer was about 1.8. At such low pH, the interference of acidic non-phosphopeptides could be reduced to a very low level. For comparison, amine-functionalized silica was used to enrich P1 at different ratios of TFA. Fig. S5b shows that the preliminarily optimal loading buffer was ACN/0.02% TFA (1/1, v/v). Aminefunctionalized silica was also used to enrich phosphopeptides from tryptic digests of ␤-casein in the optimal loading buffer. As shown in Fig. 4a, apart from three phosphopeptides, certain nonphosphopeptides also appeared in the spectrum. Increasing the TFA content seemed to be a solution, but this resulted in poor recovery of phosphopeptides (Fig. 4b and c). For instance, when the loading buffer was ACN/0.2% TFA (1/1, v/v), only one phosphopeptide was observed (Fig. 4c). The pH of loading buffer is the key factor influencing the recovery and specificity in AEX-based phosphopeptide enrichment. Lower pH can improve the specificity but reduce the recovery. An ideal AEX sorbent can enrich phosphopeptide at a pH with good balance between specificity and recovery. As nitrogen has one more electron than carbon and the atomic percentage of nitrogen is about 57%, g-C3 N4 exhibits conspicuously electron-rich (basic) surface property. Due to the incomplete condensation, the g-C3 N4 prepared by calcination of melamine contains a small amount of hydrogen existing in primary or secondary amine groups on the terminal edges. The presence of these surface defects are believed to promote the electron re-localization on the surface, which can reinforce the basic property of the surface [41]. Consequently, g-C3 N4 has high density of basic functional sites that provide a large binding capacity of phosphopeptides and show high tolerance for the acid (TFA) content in sample solution [57]. On the other hand, the

Fig. 4. MALDI mass spectra of tryptic digests of ␤-casein after enrichment by aminefunctionalized silica in different loading buffers including ACN/0.02% TFA (1/1, v/v) (a), ACN/0.1% TFA (1/1, v/v) (b), ACN/0.2% TFA (1/1, v/v) (c). The concentration of tryptic digests of ␤-casein was 1.0 × 10−7 M. The phosphopeptides are marked with “␤n”.

number of basic groups on the surface of amine-functionalized silica is limited, which make the tuning of loading condition (pH) difficult to achieve good recovery and high specificity simultaneously in phosphopeptide enrichment. In addition, the surface property of g-C3 N4 is relatively unitary and homogeneous, which can greatly decrease the nonspecific adsorption of peptides. As a result, the performance of g-C3 N4 on phosphopeptide enrichment is obviously better than that of amine-functionalized silica. 3.3. Efficiency of phosphopeptide enrichment with MCN The as-prepared MCN was directly applied to phosphopeptide enrichment. The magnetic property of MCN made the extraction

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8 6

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

Fig. 5. MALDI mass spectra obtained by direct analysis (a, c, e), or after enrichment by MCN (b, d, f) from the tryptic digest of a mixture of ␤-casein/BSA with the ratio of 1:1 (a, b), 1:10 (c, d), or 1:100 (e, f). The concentration of tryptic digests of ␤-casein was 1.0 × 10−7 M. The phosphopeptides are marked with “ ␤n”. The detailed sequence information of the identified phosphopeptides is listed in Table S1.

process convenient and fast. To evaluate the specificity, MCN was used to extract phosphopeptides from the mixtures of the tryptic digests of ␤-casein and BSA with molar ratios of 1:1, 1:10 and 1:100. Fig. 5a, c and e displays the mass spectra for the direct analysis of the mixtures. Along with the increasing ratio of BSA digestion, the signals of phosphopeptides were dramatically suppressed by non-phosphopeptides and the identification of phosphopeptides became impossible. However, as shown in Fig. 5b, d and f, five phosphopeptides could be easily detected after enrichment with MCN, even when the ratio of ␤-casein and BSA increased to 1:100. These results showed the high specificity of MCN in phosphopeptide enrichment from complex peptide mixtures. Notably, Fe3 O4 @SiO2 was also used to enrich phosphopeptides from tryptic digests of ␤casein and no assignable phosphopeptide signal could be detected

(data not shown). Therefore, the capture of phosphopeptides with MCN was based on the interaction between analytes and g-C3 N4 . The performance of MCN was further investigated by enrichment of phosphopeptides from tryptic digests of nonfat milk. As for the direct analysis, the signals of non-phosphopeptides dominated the spectrum and only five phosphopeptides were observed (Fig. 6a). Whereas, after extraction by MCN, 20 phosphopeptides were clearly identified in the spectrum with very few interferences (Fig. 6b). The detailed sequence information of the identified phosphopeptides is listed in Table S1. These results indicated that the proposed MCN had excellent selectivity toward phosphopeptides in the real in-solution digestion samples. Human serum was also used to evaluate the enrichment capability of MCN. The endogenous phosphopeptides in serum have been proved to be important biomarkers associated with some human

Fig. 6. MALDI mass spectra of tryptic digests of nonfat milk prior to enrichment (a) and after enrichment by MCN (b). MALDI mass spectra of human serum without before enrichment (c) and after enrichment by MCN (d).

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

7

Fig. 7. The number of identified phosphopeptides from tryptic digests of rat brain lysate after enrichment by MCN or TiO2 (a). Venn diagram showing the overlapping of identified phosphopeptides after enrichment by MCN or TiO2 (b).

diseases [58]. Because of the high complexity of the sample, direct analysis of serum phosphopeptides is difficult. As shown in Fig. 6c, only one phosphopeptide with poor resolution could be identified by direct analysis. After enrichment with MCN, four phosphopeptides derived from fibrinopeptide A were distinctly isolated and detected (Fig. 6d). The detailed sequence information of the four phosphopeptides is listed in Table S2. These results demonstrated that the MCN was a promising material for the enrichment of phosphopeptides from complex biological samples.

(21475098, 91217309), and the Natural Science Foundation of Hubei Province, China (2014CFA002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2016.01. 080. References

3.4. Phosphopeptide enrichment from tryptic digests of rat brain lysate MCN was further applied to enrich phosphopeptides from tryptic digests of rat brain lysate. Commercial TiO2 was also used for the comparison. As shown in Fig. 7a and Tables S3 and S4, 2576 unique phosphopeptides representing 2735 phosphorylation sites were identified by MCN, while 1236 unique phosphopeptides representing 1359 phosphorylation sites were detected by commercial TiO2 . The ratio of the number of phosphopeptides to that of all identified peptides was defined as the value of specificity and results showed that MCN exhibited higher specificity (80.8%) than that of commercial TiO2 (68.4%), hence possessed better performance in phosphopeptide enrichment than commercial TiO2 . In addition, as shown in the Venn diagram (Fig. 7b), only 503 unique phosphopeptides (15.2% of the total pool) were identified by both of the two materials, indicating that these two materials were complementary in phosphopeptide enrichment.

4. Conclusions In conclusion, magnetic graphitic carbon nitride (MCN) was successfully prepared by a simple method and served efficiently as an AEX material for specific phosphopeptide enrichment. This material had abundant binding sites on the surface and could keep excellent retention for phosphopeptides at low pH. With these benefits, MCN showed remarkable performance in capture of phosphopeptides from complex biological samples. The introduction of MCN is an improvement in AEX-based phosphopeptide enrichment. In addition, development of a new adsorbent may achieve complementary phosphoproteomics results to those of current methods. We also believe that this developed MCN has great potential in catalysis, sensors and other fields.

Acknowledgements The authors are grateful for financial support from the National Basic Research Program of China (973 Program) (2013CB910702, 2012CB720601), the National Natural Science Foundation of China

[1] R. Aebersold, M. Mann, Mass spectrometry-based proteomics, Nature 422 (2003) 198–207. [2] J. Reinders, A. Sickmann, State-of-the-art in phosphoproteomics, Proteomics 5 (2005) 4052–4061. [3] T.E. Thingholm, O.N. Jensen, M.R. Larsen, Analytical strategies for phosphoproteomics, Proteomics 9 (2009) 1451–1468. [4] D.C.H. Lee, A.R. Jones, S.J. Hubbard, Computational phosphoproteomics: from identification to localization, Proteomics 15 (2015) 950–963. [5] E. Amit, R. Obena, Y.T. Wang, R. Zhuravel, A.J.F. Reyes, S. Elbaz, D. Rotem, D. Porath, A. Friedler, Y.J. Chen, S. Yitzchaik, Integrating proteomics with electrochemistry for identifying kinase biomarkers, Chem. Sci. 6 (2015) 4756–4766. [6] B. Eyrich, A. Sickmann, R.P. Zahedi, Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies, Proteomics 11 (2011) 554–570. [7] N. Rauniyar, J.R. Yates III, Isobaric labeling-based relative quantification in shotgun proteomics, J. Proteome Res. 13 (2014) 5293–5309. [8] F. Wang, C. Song, K. Cheng, X. Jiang, M. Ye, H. Zou, Perspectives of comprehensive phosphoproteome analysis using shotgun strategy, Anal. Chem. 83 (2011) 8078–8085. [9] 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. [10] G.H. Han, M.L. Ye, H.F. Zou, Development of phosphopeptide enrichment techniques for phosphoproteome analysis, Analyst 133 (2008) 1128–1138. [11] X.S. Li, X. Chen, H. Sun, B.F. Yuan, Y.Q. Feng, Perovskite for the highly selective enrichment of phosphopeptides, J. Chromatogr. A 1376 (2015) 143–148. [12] J. Rush, A. Moritz, K.A. Lee, A. Guo, V.L. Goss, E.J. Spek, H. Zhang, X.M. Zha, R.D. Polakiewicz, M.J. Comb, Immunoaffinity profiling of tyrosine phosphorylation in cancer cells, Nat. Biotechnol. 23 (2005) 94–101. [13] J. Chen, S. Shinde, M.H. Koch, M. Eisenacher, S. Galozzi, T. Lerari, K. Barkovits, P. Subedi, R. Krueger, K. Kuhlmann, B. Sellergren, S. Helling, K. Marcus, Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies, Sci. Rep. 5 (2015) 11438. [14] W.A. Tao, B. Wollscheid, R. O’Brien, J.K. Eng, X.J. Li, B. Bodenmiller, J.D. Watts, L. Hood, R. Aebersold, Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry, Nat. Methods 2 (2005) 591–598. [15] P. van der Veken, E.H.C. Dirksen, E. Ruijter, R.C. Elgersma, A.J.R. Heck, D.T.S. Rijkers, M. Slijper, R.M.J. Liskamp, Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides, Chembiochem 6 (2005) 2271–2280. [16] K. Engholm-Keller, M.R. Larsen, Technologies and challenges in large-scale phosphoproteomics, Proteomics 13 (2013) 910–931. [17] C.L. Nilsson, Advances in quantitative phosphoproteomics, Anal. Chem. 84 (2012) 735–746. [18] L. Zhao, H.Q. Qin, Z.Y. Hu, Y. Zhang, R.A. Wu, H.F. Zou, A poly(ethylene glycol)-brush decorated magnetic polymer for highly specific enrichment of phosphopeptides, Chem. Sci. 3 (2012) 2828–2838. [19] X.S. Li, J.H. Wu, Y. Zhao, W.P. Zhang, Q. Gao, L. Guo, B.F. Yuan, Y.Q. Feng, Preparation of magnetic polymer material with phosphate group and its

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080

G Model CHROMA-357281; No. of Pages 8

ARTICLE IN PRESS G.-T. Zhu et al. / J. Chromatogr. A xxx (2016) xxx–xxx

8

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

application to the enrichment of phosphopeptides, J. Chromatogr. A 1218 (2011) 3845–3853. Y. Yan, Z. Zheng, C. Deng, Y. Li, X. Zhang, P. Yang, Hydrophilic polydopamine-coated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis, Anal. Chem. 85 (2013) 8483–8487. A. Leitner, Phosphopeptide enrichment using metal oxide affinity chromatography, TrAC—Trends Anal. Chem. 29 (2010) 177–185. J.H. Wu, X.S. Li, Y. Zhao, W. Zhang, L. Guo, Y.Q. Feng, Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings, J. Chromatogr. A 1218 (2011) 2944–2953. J.H. Wu, X.S. Li, Y. Zhao, Q. Gao, L. Guo, Y.Q. Feng, Titania coated magnetic mesoporous hollow silica microspheres: fabrication and application to selective enrichment of phosphopeptides, Chem. Commun. 46 (2010) 9031–9033. M. Zarei, A. Sprenger, F. Metzger, C. Gretzmeier, J. Dengjel, Comparison of ERLIC-TiO2 , HILIC-TiO2 , and SCX-TiO2 for global phosphoproteomics approaches, J. Proteome Res. 10 (2011) 3474–3483. G. Han, M. Ye, H. Zhou, X. Jiang, S. Feng, X. Jiang, R. Tian, D. Wan, H. Zou, J. Gu, Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography, Proteomics 8 (2008) 1346–1361. A. Motoyama, T. Xu, C.I. Ruse, J.A. Wohlschlegel, J.R. Yates III, Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides, Anal. Chem. 79 (2007) 3623–3634. C.X. Yang, X.F. Zhong, L.J. Li, Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics, Electrophoresis 35 (2014) 3418–3429. M.M. Dong, M.H. Wu, F.J. Wang, H.Q. Qin, G.H. Han, J. Dong, R.A. Wu, M.L. Ye, Z. Liu, H.F. Zou, Coupling strong anion-exchange monolithic capillary with MALDI-TOF MS for sensitive detection of phosphopeptides in protein digest, Anal. Chem. 82 (2010) 2907–2915. A.J. Alpert, O. Hudecz, K. Mechtler, Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography, Anal. Chem. 87 (2015) 4704–4711. M.L. Hennrich, V. Groenewold, G.J.P.L. Kops, A.J.R. Heck, S. Mohammed, Improving depth in phosphoproteomics by using a strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach, Anal. Chem. 83 (2011) 7137–7143. M.S. Ritorto, K. Cook, K. Tyagi, P.G.A. Pedrioli, M. Trost, Hydrophilic strong anion exchange (hSAX) chromatography for highly orthogonal peptide separation of complex proteomes, J. Proteome Res. 12 (2013) 2449–2457. S.B. Ficarro, Y. Zhang, M.J. Carrasco-Alfonso, B. Garg, G. Adelmant, J.T. Webber, C.J. Luckey, J.A. Marto, Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis, Mol. Cell. Proteomics 10 (2011), O111.011064. T.S. Nuhse, A. Stensballe, O.N. Jensen, S.C. Peck, Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry, Mol. Cell. Proteomics 2 (2003) 1234–1243. S. Nie, J. Dai, Z.B. Ning, X.J. Cao, Q.H. Sheng, R. Zeng, Comprehensive profiling of phosphopeptides based on anion exchange followed by flow-through enrichment with titanium dioxide (AFET), J. Proteome Res. 9 (2010) 4585–4594. M. Atakay, Ö. C¸elikbıc¸ak, B. Salih, Amine-functionalized sol–gel-based lab-in-a-pipet-tip approach for the fast enrichment and specific purification of phosphopeptides in MALDI-MS applications, Anal. Chem. 84 (2012) 2713–2720. F. Goettmann, A. Fischer, M. Antonietti, A. Thomas, Chemical synthesis of mesoporous carbon nitrides using hard templates and their use as a metal-free catalyst for Friedel–Crafts reaction of benzene, Angew. Chem. Int. Ed. Engl. 45 (2006) 4467–4471. P. Xiao, Y. Zhao, T. Wang, Y. Zhan, H. Wang, J. Li, A. Thomas, J. Zhu, Polymeric carbon nitride/mesoporous silica composites as catalyst support for Au and Pt nanoparticles, Chem. Eur. J. 20 (2014) 2872–2878. M. Groenewolt, M. Antonietti, Synthesis of g-C3 N4 nanoparticles in mesoporous silica host matrices, Adv. Mater. 17 (2005) 1789–1792.

[39] Y. Wang, X.C. Wang, M. Antonietti, Polymeric graphitic carbon nitride as a heterogeneous organocatalyst: from photochemistry to multipurpose catalysis to sustainable chemistry, Angew. Chem. Int. Ed. 51 (2012) 68–89. [40] Y.T. Gong, M.M. Li, H.R. Li, Y. Wang, Graphitic carbon nitride polymers: promising catalysts or catalyst supports for heterogeneous oxidation and hydrogenation, Green Chem. 17 (2015) 715–736. [41] J. Zhu, P. Xiao, H. Li, S.A. Carabineiro, Graphitic carbon nitride: synthesis, properties, and applications in catalysis, ACS Appl. Mater. Interfaces 6 (2014) 16449–16465. [42] S.W. Cao, J.X. Low, J.G. Yu, M. Jaroniec, Polymeric photocatalysts based on graphitic carbon nitride, Adv. Mater. 27 (2015) 2150–2176. [43] A. Thomas, A. Fischer, F. Goettmann, M. Antonietti, J.O. Muller, R. Schlogl, J.M. Carlsson, Graphitic carbon nitride materials: variation of structure and morphology and their use as metal-free catalysts, J. Mater. Chem. 18 (2008) 4893–4908. [44] F. Su, M. Antonietti, X. Wang, mpg-C3 N4 as a solid base catalyst for Knoevenagel condensations and transesterification reactions, Catal. Sci. Technol. 2 (2012) 1005–1009. [45] Z. Lin, J. Zheng, G. Lin, Z. Tang, X. Yang, Z. Cai, Negative ion laser desorption/ionization time-of-flight mass spectrometric analysis of small molecules using graphitic carbon nitride nanosheet matrix, Anal. Chem. 87 (2015) 8005–8012. [46] H.B. Zheng, J. Ding, S.J. Zheng, G.T. Zhu, B.F. Yuan, Y.Q. Feng, Facile synthesis of magnetic carbon nitride nanosheets and its application in magnetic solid phase extraction for polycyclic aromatic hydrocarbons in edible oil samples, Talanta 148 (2016) 46–53. [47] S. Kumar, S. Tonda, B. Kumar, A. Baruah, V. Shanker, Synthesis of magnetically separable and recyclable g-C3 N4 –Fe3 O4 hybrid nanocomposites with enhanced photocatalytic performance under visible-light irradiation, J. Phys. Chem. C 117 (2013) 26135–26143. [48] M. Wang, S.H. Cui, X.D. Yang, W.T. Bi, Synthesis of g-C3 N4 /Fe3 O4 nanocomposites and application as a new sorbent for solid phase extraction of polycyclic aromatic hydrocarbons in water samples, Talanta 132 (2015) 922–928. [49] M. Wang, X. Yang, W. Bi, Application of magnetic graphitic carbon nitride nanocomposites for the solid-phase extraction of phthalate esters in water samples, J. Sep. Sci. 38 (2015) 445–452. [50] S.N. Xu, Q. Zhao, H.B. He, B.F. Yuan, Y.Q. Feng, Q.W. Yu, Rapid determination of polycyclic aromatic hydrocarbons in environmental water based on magnetite nanoparticles/polypyrrole magnetic solid-phase extraction, Anal. Methods 6 (2014) 7046–7053. [51] G.T. Zhu, X.S. Li, Q. Gao, N.W. Zhao, B.F. Yuan, Y.Q. Feng, Pseudomorphic synthesis of monodisperse magnetic mesoporous silica microspheres for selective enrichment of endogenous peptides, J. Chromatogr. A 1224 (2012) 11–18. [52] S.C. Yan, Z.S. Li, Z.G. Zou, Photodegradation performance of g-C3 N4 fabricated by directly heating melamine, Langmuir 25 (2009) 10397–10401. [53] Y.G. Wang, F. Wang, Y.H. Zuo, X.D. Zhang, L.F. Cui, Simple synthesis of ordered cubic mesoporous graphitic carbon nitride by chemical vapor deposition method using melamine, Mater. Lett. 136 (2014) 271–273. [54] M.J. Bojdys, J.O. Müller, M. Antonietti, A. Thomas, Ionothermal synthesis of crystalline condensed, graphitic carbon nitride, Chem. Eur. J. 14 (2008) 8177–8182. [55] N. Xu, Y. Wang, M. Rong, Z. Ye, Z. Deng, X. Chen, Facile preparation and applications of graphitic carbon nitride coating in solid-phase microextraction, J. Chromatogr. A 1364 (2014) 53–58. [56] S. Lu, Q. Luo, X.C. Li, J.H. Wu, J.N. Liu, S.X. Xiong, Y.Q. Feng, F.Y. Wang, Inhibitor screening of protein kinases using MALDI-TOF MS combined with separation and enrichment of phosphopeptides by TiO2 nanoparticle deposited capillary column, Analyst 135 (2010) 2858–2863. [57] Y.J. Zhang, A. Thomas, M. Antonietti, X.C. Wang, Activation of carbon nitride solids by protonation: morphology changes, enhanced ionic conductivity, and photoconduction experiments, J. Am. Chem. Soc. 131 (2009) 50–51. [58] L.H. Hu, H.J. Zhou, Y.H. Li, S.T. Sun, L.H. Guo, M.L. Ye, X.F. Tian, J.R. Gu, S.L. Yang, H.F. Zou, Profiling of endogenous serum phosphorylated peptides by titanium(IV) immobilized mesoporous silica particles enrichment and MALDI-TOFMS detection, Anal. Chem. 81 (2009) 94–104.

Please cite this article in press as: G.-T. Zhu, et al., Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.01.080