Bis(zinc(II)-dipicolylamine)-functionalized sub-2 μm core-shell microspheres for the analysis of N-phosphoproteome

Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 μm core-shell silica microspheres (SiO2@DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.

The main reason that hinders the study of N-pho is derived from the intrinsic lability of P-N bond. Since the phosphorus dorbital of π-bonding is in a higher energy shell, the overlap between nitrogen lone pair orbital and the phosphoryl π-bond is scarce. Therefore, the P-N bond is not benefited from the stabilization caused by electronic delocalization. In addition, the basic of nitrogen is preserved, which markedly enhances the leaving ability of the amino group once protonated 19,20 .
In modern biological approaches, studies were mainly focused on finding target proteins such as kinases and phosphatases. NME1 and NME2 are the only two mammalian protein histidine kinases reported so far 21 , and LHPP is discovered as the protein histidine phosphatase and tumor suppressor 22 . Likewise, McsB/ YwIE is the well-known arginine kinase/phosphatase pair 23 . However, neither pLys kinases nor phosphatases were found so far, and histone H1 was the only reported pLys protein 24 .
To globally elucidate one kind of N-pho, much effort has been made in preparing effective antibodies. Nevertheless, the acid instability and structural flexibility of N-pho makes it difficult to induce immunization by classical methods. Recently, benefited from stable pHis and pArg analogs, several antibodies have been prepared 25,26 . Fuhs et al. 27 generated the first pHis monoclonal antibody, and some pHis candidates related to cell cycle functions were discovered in mammalian cells. Fuhrmann et al. 28 developed two isosteres of pArg and generated the first high-affinity pan-pArg antibody for screening the chaperone proteins, ClpC and GroEL in B. subtilis DywlE strain. Hunter group identified 425 Npho sites from HeLa lysates using hydroxyapatite and 1, 3-pHis monoclonal antibodies (HAP/pHis mAbs) 29 . However, the structural multiformity introduced by adding phosphate groups to diverse residues makes it rather difficult to develop highly efficient pan-specific antibodies for the global study of Nphosphoproteome. Besides, specific N-pho variants are usually predisposed by certain antibodies and thus can cause biases. For N-phosphoproteome analysis, antibody-independent methods can solve the above problems. However, due to the instability of P-N bond under acidic conditions, the currently used enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides, resulting that the landscape of N-phosphoproteome was still covered 30,31 . Inspired by naturally occurring phosphatases, which provide specific Zn(II)-central enzymatic pockets to bind phosphate units of substrates, bis(zinc(II)-dipicolylamine) molecular (DpaZn) is designed for phosphate targets recognition under neutral conditions 32 . Moreover, phos-Tag beads with Zn (II) were used for enrichment of non-canonical phosphorylation peptides 33,34 , which further demonstrated the reliability of 2Zn (II). Therefore, DpaZn molecular functionalized materials show great potential for N-phosphopeptides enrichment under neutral conditions.
In this work, DpaZn-functionalized sub-2-μm core-shell silica microspheres (SiO 2 @DpaZn) are designed for on-tip N-phosphopeptides enrichment under neutral conditions. A total of 27 N-pho sites are identified from Escherichia coli. In addition, SiO 2 @DpaZn are applied for the analysis of mammalian Nphosphopeptides. In total, 3384 N-pho sites, containing 611 pHis, 1618 pLys, and 1155 pArg, are identified from HeLa cell lysates. We provide reliable technical support for N-pho studies.

Results
Design and evaluation of bis(zinc(II)-dipicolylamine) functionalized sub-2-μm core-shell SiO 2 microspheres. Theoretically under neutral conditions, 2Zn(II)-chelated bis (dipicolylamine) (Dpa) groups, terms DpaZn, could bind with phosphorylated target (including both N-phosphorylated and O-phosphorylated targets) and form the stable 1:1 complex 32 . Each Zn(II) ion is coordinated with two pyridyl nitrogen atoms, a tertiary amine, a phenoxy anion, and an oxygen atom from phosphate group (Fig. 2a inset). Herein, nuclear magnetic resonance (NMR) was applied to evaluate whether DpaZn could recognize Nphosphorylated target in neutral solution. As shown in Supplementary Fig. 1a, in 1 H-NMR, two peaks corresponding to the two protons on imidazole regions of pHis were observed. Upon the addition of DpaZn, such two signals were upfield-shifted by around 0.1 p.p.m. In 31 P-NMR study, the same upfield shift of phosphorus was also observed ( Supplementary Fig. 1b), which validated the recognition between DpaZn and the phosphate group.
Moreover, it is noticed that the P-N bond is also heat-labile even under neutral conditions 35 Therefore, rapid enrichment is preferred to improve the recovery of N-phosphopeptides 36 . Herein, sub-2-μm core-shell SiO 2 microspheres were designed as the substrate, not only to achieve the specific enrichment within a shorter time, contributed by the fast mass transfer, but also to provide adequate reaction sites for DpaZn immobilization, ensured by the large specific surface area. Accordingly, we could expect that the combination of DpaZn functional groups and core-shell silica microspheres might be effective for the capture of N-phosphoproteome. SiO 2 @DpaZn were prepared by seed growth and template growth methods according to our previous work 37 , followed by carboxyl terminal Dpa immobilization and two Zn(II) ions coordination ( Fig. 2a and Supplementary Fig. 2). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed the highly spherical microspheres with radial core-shell structure, with the core diameter of ∼1.3 μm and the shell thickness of ∼0.16 μm (Fig. 2b, c). The peaks of Zn2p confirmed the existence of Zn(II) ions on the microspheres ( Fig. 2d and Supplementary Fig. 3). The surface area and pore size of SiO 2 @DpaZn were calculated to be 83.2 m 2 /g and 17.4 nm, respectively (Fig. 2e, f). The content of DpaZn was calculated as about 14.3% wt (∼143 mg/g) by thermogravimetric analysis (TGA) analysis (Fig. 2g). The above results demonstrated the successful preparation of the material.
The enrichment ability of SiO 2 @DpaZn toward N-phosphopeptide was evaluated by capturing pHis peptide TS p HYSIMAR from BSA digests (1:100, m/m) under neutral condition (workflow shown in Supplementary Fig. 4). The peaks of N-phosphopeptide and its dephosphorylated counterpart could hardly be observed by direct analysis (Fig. 3a). However, after enrichment, most nonphosphopeptides were removed and N-phosphopeptide with enhanced intensity dominated the MS spectra (Fig. 3b), indicating the high enrichment selectivity of SiO 2 @DpaZn.
Establishment of on-tip enrichment strategy. Commonly in solution enrichment, long incubation time and tedious operation, including vortex and ultrasound, are usually required to ensure the enrichment efficiency. However, the N-pho was rapidly hydrolyzed to 73.2% and 40.1% in these processes within 10 min, respectively (Fig. 3c). Therefore, to improve the recovery of Nphosphopeptides, the entire on-tip enrichment was achieved within 30 min since the adsorption equilibrium by SiO 2 @DpaZn could be reached within 5 min (Fig. 3d). By quantitative proteomics ( Supplementary Fig. 5), the recovery of Nphosphopeptide by on-tip enrichment was fourfold to that of in-solution enrichment, whereas the total time was shortened to 1/3 ( Supplementary Fig. 6), beneficial to achieve the deepcoverage N-phosphoproteome. On-tip method also increased the   Identification of N-phosphorylated peptides from E. coli lysates. Encouraged by the above results, the comprehensive Nphosphoproteome of Luria-Bertani-cultured E. coli was performed after the enrichment by SiO 2 @DpaZn-based on-tip strategy. Herein, peptide-spectrum matches (PSMs) were strictly checked according to the rules shown in Supplementary Fig. 7. With FDR at peptide and PSMs level set <1%, and the cut-off value of ion score as 20, combined with manual site localization, the direct evidence of 27 N-pho sites (15 pHis, 8 pLys, and 4 pArg) and 12 O-pho sites (Supplementary Dataset 1) was identified after removing the pLys/ pArg peptides localized to the C-terminus. Furthermore, 3 pHis sites (PPS H421, manX H20, and ptsI H189) were in accordance with the previous method 39 . To further explore the influence of different carbon sources on N-pho sites in E. coli, Nphosphopeptides from M9 minimal medium-cultured E. coli (glucose or glycerol) were enriched by our method. After removing the pLys/pArg peptides localized to the C-terminus, 99 N-pho sites (19 pHis, 38 pLys, and 42 pArg) and 101 O-pho sites (39 pSer, 56 pThr, and 6 pTyr) with localization probability over 0.75 were identified (Supplementary Dataset 2). These results demonstrated the reliability of our method to N-phosphoproteome analysis.
To further confirm the authenticity of the N-pho sites identified in our study, seven of the N-phosphopeptides were synthesized (Supplementary Dataset 1). The MS/MS spectrum and retention time of the synthetic peptides were compared with that obtained from in vivo peptides. As an example, autonomous glycyl radical cofactor (YfiD) is documented as an acid-inducible protein in E. coli in response to environmental stress 40 , and has been reported to be a phosphoprotein with the phosphoresidue(s) unstable to acid 41 . However, the exact modified sites were still unknown. In this study, two N-pho sites (H26 and K48) on YfiD were identified (AEAGIVISAS p HNPFYDNGIK and AGYAEDEVVAVS p KLGDIEYR). The MS/MS spectra of the peptides from in vivo (AEAGIVISAS +79.98Da HNPFYDNGIK and AGYAEDEVVAVS +79.98 Da KLGDIEYR) with a mass shift of +79.98 Da at the histidine and lysine residues had the same MS/ MS spectra as that of the synthetic peptides with a phosphate group on histidine and lysine, respectively (AEAGIVISAS p HNP-FYDNGIK and AGYAEDEVVAVS p KLGDIEYR) (Fig. 4a, b). In addition, the two synthetic N-pho peptides eluted at the same time with the in vivo peptides on HPLC (Fig. 5a, b), confirming that the detected mass shift of +79.98 Da in the in vivo-derived peptides was caused by N-pho. Similarly, the MS/MS spectra and retention time of other N-phosphopeptides were also examined and exhibited in Supplementary Fig. 8. Taken together, we confirmed the authenticity of these reported N-phosphopeptides. Moreover, after label-free quantification by the MaxQuant software, calculated Pearson correlation coefficients (PCCs) for the quantified peptides in triplicate (each replicate corresponding to a different enrichment and different LC-MS/MS injection) were all around 0.90 (n = 3, Supplementary Fig. 9), demonstrating our enrichment method is robust and reproducible.
Identification of N-phosphorylated peptides from HeLa lysates.  27 . However, we did not identify any known pHis sites including NME1/2 1-pHis 118, PGAM 3-pHis11, and NDK1 1-pH117 from biological samples. Therefore, to be cautious, we stated that we did not know whether our method enriches both forms of pHis from complex biological samples. Motif analysis is conducive to evaluate the features of the    N-phosphoproteins in HeLa cells, and the ±10 residue sequence windows were generated from all N-pho sites and tested against HeLa proteome background. Consistent with the previous studies of pHis, the data exhibited enrichment of leucine residue around the N-pho sites ( Supplementary Fig. 10a). This could either be a preference for the His kinase, or alternatively this could be because peptides with leucine residues in the vicinity of the Npho residue could be more resistant to hydrolysis. The result was further verified in HEPG2 cells ( Supplementary Fig. 10b). To elucidate the biological function of N-phosphoproteins identified from HeLa cells, gene ontology (GO) analysis of the identified 2596 N-phosphoproteins was performed, which revealed the significant enrichment of biological process terms of metabolism, regulation of metabolism, organization, and immune response ( Supplementary Fig. 11). Besides, we also found that these proteins participated in ATP binding, ATPase activity, RNA binding, nucleotide binding, and protein kinase binding, which was consistent with previous results for HeLa cells 29,42 .
Investigation of the interaction between SiO 2 @DpaZn and Npho peptides. We made further effort to elucidate the interaction between SiO 2 @DpaZn and N-pho under neutral conditions. As shown in Fig. 6a, the enrichment selectivity of TS p HYSIMAR by various materials is in the order of SiO 2 @DpaZn > SiO 2 @Dpa > SiO 2 @NH 2 , indicating that metal ion chelation makes the main contribution to the selectivity, and further enhanced by hydrophilic interaction (Supplementary Fig. 12). We made further confirmation of such deduction by adjusting the mixed ratio of ACN and NH 3 ·H 2 O respectively mixed with the N-phosphopetide, since ACN facilitated the hydrophilic interaction, whereas NH 3 ·H 2 O inhibited the chelation between Zn(II) and phosphate groups. As shown in Fig. 6b, with the decrease of ACN and the increase of NH 3 ·H 2 O, non-phosphopeptides, mono-phosphopeptides (m/z 1145, 2065, and 2556,) and the multi-phosphopeptide (m/z 3122) were eluted in sequence, in accordance with our expectation. Moreover, we found the zeta potential of SiO 2 @DpaZn kept stable at about +40 mV around pH 7.0 (Fig. 6c), enabling the additional electrostatic interaction with phosphate groups. Collectively, in the recognition process of phosphate groups, the primary coordination bonding with DpaZn was strengthened by additional secondary noncovalent interactions including hydrophilic and electrostatic interactions. To further quantify the interaction between SiO 2 @DpaZn and N-phosphopeptides, several in vivo-derived N-phosphopeptides from E. coli with different net charges were synthesized and applied to isothermal titration calorimetry (ITC) experiments. The interaction between DpaZn and N-phosphopeptides was endothermic, indicating it an entropy-driven process ( Supplementary Fig. 13)  N-phosphopeptides ranged from 13.14 μM to 113.64 μM (Table 1). In the ITC experiments, DpaZn was uniformly distributed in the solution, which was actually inconsistent with the actual on-tip enrichment. Just like the immobilized enzymatic reactor can condense the concentration of enzyme and accelerate digestion 38 , SiO 2 @DpaZn was tightly packed into a micropipette tip to achieve a volume of about 3.8 μL for cell lysates enrichment. Then the actual concentrate of DpaZn in the enrichment system could be calculated as 130.9 mM. From the perspective of kinetics, high concentration DpaZn is conducive for the rapid recognition of low-abundance Npho peptides.

Discussion
In this study, SiO 2 @DpaZn was carefully designed for on-tip enrichment of N-phosphopeptides. In fact, from the identification results of E. coli and HeLa, O-pho peptides were inevitably enriched at the same time. Indeed, SiO 2 @DpaZn can bind both N-pho peptides and O-pho peptides. However, according to previous papers 43,44 , the DpaZn coordination complex has a vacancy on each Zn(II) ion that the phosphate anion [RO(N) PO 3 ] can access to form RO(N)PO 3 -DpaZn complex. Compared with P-O bond, the N atom of P-N bond has a stronger electron donating ability than the O atom, which makes the structure of RNPO 3 -DpaZn more stable. Therefore, this might result in Npho peptides being preferentially enriched over O-pho peptides under the neutral conditions. This also explained the relatively high proportion of N-pho peptides in our results.
Due to the important biological function of N-pho, several groups are committed to develop antibody-independent methods to enrich N-pho peptides. Potel et al. reported a Fe 3+ -IMAC-     Table 2). Compared with our results, no identical pHis site was found, which might be attributed to different enrichment conditions. However, considering that pLys and pArg suffered from severe hydrolysis under mild acidic conditions or long-term operation 46,47 , our method might be more suitable for comprehensive N-pho sites discovery. Furthermore, 27 unphosphorylated peptides in our results were identified as pHis peptides by Potel et al., which might be attributed to acidic separation. Although acidic separation contributes to good separation, it might also increase the risk of N-pho hydrolysis. To overcome the problem, photonic crystals with ultra-efficiency separation might be an alternative to significantly reduce separation time 48 .
In additional, a strong anion exchange-based method (SAX) was developed by Hardman et al. to reveal human non-canonical phosphorylation. A total of 781 unique N-pho sites, including 225 pHis, 278 pLys, and 278 pArg, were identified from HeLa lysates ( Table 2). Comparing HeLa N-pho results between the existing three methods, low overlap of N-pho sites was found ( Supplementary Fig. 14), which might be due to different enrichment mechanisms under neutral conditions and different separation and MS conditions. However, these methods had meaningful complementarities which were beneficial for N-pho sites identification. For example, an interesting result was found about the pArg sites on protein SRRM2. Four pArg sites (R294, R320, R986, and R2103) were found by both SiO 2 @DpaZn and SAX. Moreover, 3 (R1494, R2131, and R2396), 6 (R302, R356, R851, R1530, R2119, and R2286) and 1 (R1879) pArg sites were exclusively identified by SAX, SiO 2 @DpaZn, and HAP/pHis mAbs, respectively. pHis is physiologically important for both bacteria and mammals [49][50][51][52] . After re-examining our data, some vital N-pho proteins that perform protein kinases or phosphatases were identified, such as Phosphoenolpyruvate synthase and Phosphoenolpyruvateprotein phosphotransferase. Although several known pHis sites were successfully identified in our study, we failed to find some known p-His sites, including NME1/2 1-pHis118, NDK1 1-pHis 117, and PGAM 3-pHis11. The similar phenomenon was found in a previous report 42,45 . It deserved in-depth analysis, and there were three possible reasons for explaining failure to identification of known pHis sites: (i) The enrichment of N-pho is carried out under neutral conditions, and so negative charge peptides would be co-eluted with N-pho peptides in this case, which might inhibit N-pho peptides identification. (ii) Some tryptic pHis-containing peptides like the histone H4 (H18) and ACLY (H760) peptides are too short or too long to be considered by MS using current parameters 29 . (iii) Acid separation conditions would increase the risk of pHis hydrolysis 29 .
In summary, the above results reveal four appealing features of SiO 2 @DpaZn: specific complexation with N-pho peptides driven by coordinate interaction at moderate binding ability, rapid adsorption within 10 min enabled by core-shell structure, high recovery and modification preservation achieved by on-tip analysis, as well as excellent enrichment ability toward diverse N-pho peptides (especially rare pLys and pArg peptides) from biological samples. A large amount of unique N-pho sites have been identified from E. coli and HeLa lysates benefiting from these different characteristics from traditional TiO 2 or IMAC materials. High discovery rates for N-pho sites and non-discrimination of recognizing all kinds of phosphorylated peptides illustrate the great potential of our material in comprehensive Nphosphoproteome analysis. Therefore, this universal method might greatly promote the studies of the world of Nphosphoproteins. In addition, the smart core-shell structure design and fast on-tip enrichment concept can be extended to other protein PTMs such as polyphosphorylation 53 and Ssulphenylation 54 , which play vital roles in biological processes but are all fragile under certain conditions and cannot be efficiently captured by artificial materials so far. By precise designing the recognition groups, controlling the enrichment conditions, together with applying the fast on-tip enrichment, these challenges might be addressed. Furthermore, if the enrichment pH value was reduced to 2.3, SiO 2 @DpaZn may be suitable for largescale pHis analysis. We believed that our core-shell structure design and fast on-tip enrichment concept would gain more attention in PTM proteomes analysis in the near future.
Characterizations. Hydrogen NMR ( 1 H-NMR) was measured by a bruker AVANCE III HD 400 MHz spectrometer at room temperature (Daltonios, Germany). Chemical shifts were recorded in parts per million (p.p.m.) using residual solvent peaks as internal references [CDCl 3 δ: 7.26 (1H)]. Molecular mass was detected by orbitrap LTQ (Thermo, USA). TEM images were collected on a transmission electron microscope operated at 120 kV (JEM-2000EX, JEOL, Tokyo, Japan). SEM images were collected on a Zeiss Merlin FEG-SEM instrument. TGA was performed under an air atmosphere with a heating rate of 10°C/min using a Netzsch STA449F3 thermogravimetric analyzer (Netzsch, Bavaria, Germany). Zeta potential was measured using the Nano-ZS90 zetasizer (Malvern, UK). X-ray photoelectron spectroscopy (XPS) characterization was carried out on an Thermo ESCALAB250Xi spectrometer with Al Kα radiation as the X-ray source (Thermo, Waltham, USA). The nitrogen adsorption and desorption isotherms were measured by QuadrasorbSI (Quadraorb, Wisconsin, USA). The Brunauer-Emmett-Teller (BET) method was used to calculate the specific surface areas with adsorption data in a relative pressure range from 0.049 to 0.252.  Sites localization probability over 0.75 is applied for all data.
Synthesis of Dpa (1). According to the literature 55 , 2,2′-dipicolylamine (3.37 g, 16.9 mmol) and paraformaldehyde (0.813 g, 27.1 mmol) solution (water/i-PrOH = 5:3 (v/v), 48 mL) was adjusted to pH 8.0 by 1 N HCl aq. After stirring at 80°C for 30 min, Boc-L-tyrosine-OMe (2.0 g, 6.77 mmol) was added, and the mixture was refluxed at 110°C for 13 h. Then, the mixture was cooled to room temperature, and i-PrOH was removed by evaporation. After cooling on an ice-bath, the solution was removed by decantation, and the precipitated viscous oil was dissolved in 50 mL of AcOEt. The solution was washed with saturated NaHCO 3 and brine followed by drying over Na 2 SO 4 . After removal of the solvent in vacuo, the residue was purified by column chromatography (SiO 2 , CH 2 Cl 2 /MeOH/ TEAB aq = 30/1/0.1 (v/v/v)) to give 1 (3.30 g, 68%) as a yellow oil. 1 Figs. 15 and 16). (2). In a typical reaction, TFA (20 mL) was added dropwise to a stirring solution of 1 (3.30 g, 4.60 mmol) in anhydrous CH 2 Cl 2 (20 mL) on ice-bath, and the solution was stirred at room temperature for 2 h. After concentration in vacuo, the residue was dissolved in water and alkalized with ammonium hydroxide solution on ice-bath. The resulting mixture was extracted with CH 2 Cl 2 . The combined organic layers were washed with brine and dried over Na 2 SO 4 . After removal of the solvent in vacuo, 2 (2.86 g, 97%) was obtained as a pale viscous oil. 1 (3). In a typtical reaction, glutaric anhydride (0.635 g, 5.57 mmol) was added to a solution of 2 (2.86 g, 4.64 mmol) in anhydrous CH 2 Cl 2 (110 mL). The mixture was stirred and refluxed at 50°C overnight. After removing the solvent by evaporation, 3 was obtained as a light yellow viscous oil. Preparation of non-porous silica. Non-porous microspheres were prepared by a seed-growth approach 56 . It contains three steps. Firstly, hydrolysis solution, containing 6.7 mL of NH 3 ·H 2 O, 5.1 mL of H 2 O, and 70 mL of CH 3 CH 2 OH, was placed in water bath at 22°C, and 4.0 mL of TEOS was added. The mixtures were reacted for 40 min under constant mechanical stirring. After the temperature was increased to 55°C, 0.64 mL of H 2 O and 4.0 mL of pre-heated TEOS were added to the mixtures and reacted for 40 min (denoted as growth process), and the growth process was repeated another three times. The obtained suspension was denoted as seeds and divided into four pieces. Secondly, we used 1 piece seed replaced equal volume hydrolysis solution, and hydrolysis solution containing seed was heated to 55°C, and the growth process was repeated three times. Afterward, the hydrolysis solution was added to the mixtures, and growth process was repeated four times. The obtained particles were centrifuged at 4000 × g for 5 min, with 95% (v/v%) CH 3 CH 2 OH and H 2 O. The obtained particles were divided to four pieces which were denoted as large particles. Finally, one piece of large particle was re-dispersed in the hydrolysis solution at 55°C, and the growth process was repeated three times. Non-porous silica was obtained by centrifugation at 3300 × g for 3 min using 95% (v/v%) CH 3 CH 2 OH and H 2 O.

Synthesis of carboxyl terminal Dpa
Preparation of sub-2-μm core-shell silica. Firstly, 1.0 g of above non-porous silica with were added to the growth system which included 100 mL of H 2 O, 5.8 mL of tridecane, and 1.0 g of CTAC. Then, 26 mg of NH 4 F and 6.0 mL of NH 3 ·H 2 O were added and the mixtures were carried out for 24 h at 90°C under stirring. The product was obtained by centrifugation at 3300 × g for 3 min. Finally, the produce was dried at 65°C for 2 h and calcined with a Ceramic Fibre Muffle Furnace (Michem, Beijing, China) at 550°C for 6 h in air. The heating procedure was from 25 to 550°C at a ramp rate of 1°C/min. The pore size was enlarged by etching in 5 M HCl at 120°C for 12 h.
Amino-functionalized sub-2-μm superficially porous silica. In a typical reaction, 0.6 g of sub-2-μm superficially porous silica particles and 2.21 mL of APTES were added to 30 mL of anhydrous toluene, and the mixtures were stirred and refluxed at 110°C for 24 h. The obtained particles were centrifuged at 4000 × g for 5 min and sequentially washed with toluene, CH 3 OH, and CH 3 COCH 3 . Adsorption kinetics of SiO 2 @DpaZn. A total of 0.4 mg of SiO 2 @DpaZn was incubated with 1.6 mL of 80% ACN (20 mM HEPES, pH 7.7) containing 10 μg of O-phosphopeptide GK8 at room temperature. After incubation for 2, 5, 10, 15, 20, and 30 min, respectively, 40 μL of the mixture was centrifuged, and the concentration of GK8 was measured by UV analysis. The adsorption capacity (Q) was calculated as follows: Q = (C 0 −C) V/m, where m is the mass (mg) of SiO 2 @DpaZn, V (mL) the volume of incubation, C 0 (mg/mL) and C (mg/mL) the concentrations of GK8 in the initial solution and the supernatant after the adsorption, respectively. Three sets of parallel experiments were conducted simultaneously.
Preparation of protein extracts from E. coli. E. coli (strain K12) was cultured in Luria-Bertani medium (LB, 5 g/L yeast extracts, 10 g/L NaCl, and 10 g/L tryptone) at 37°C overnight or in M9 minimal medium (consisting of 6 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl, supplemented with either additional 0.5% (w/ v) glucose or 0.5% glycerol) at 37°C to exponential phase and stationary phase, and were harvested by centrifugation at 4000 × g for 2 min at 4°C. Cells were washed three times with cold PBS and resuspended in lysis buffer (8 M urea, 10 mM PBS, 1% (v/v) protease inhibitor cocktail, 10 mM EDTA, and 1% (v/v) phosphatase inhibitor cocktail). After ultrasonication for 120 s (20 s interval every 10 s) in an ice bath, insoluble portions were separated from the soluble ones by centrifugation at 16,000 × g for 30 min at 4°C, and protein concentrations were determined using BCA assay. to 80% B, and 10 min to kept at 80% B). A Orbitrap Fusion Lumos mass spectrometer (Thermo-Fisher, San Jose, CA, USA) was operated in full scan (60,000 FWHM, 350-1500 m/z) and product scan (15,000 FWHM, 100-1000 m/z) modes at positive ion mode with orbitrap detector. The electro-spray voltage was 2.3 kV, and the heated capillary temperature was 320°C. All the mass spectra were recorded with Xcalibur software (version 3.1, Thermo Fisher Scientific, USA). MS/MS spectra were acquired by data-dependent acquisition mode, and total cycle time was set to 3 s. Peptides with charge state ≥2 were selected for sequencing in the HCD collision cell with collision energy of 30%. HPLC separation for Hela lysate is as same as that for E. coli cultured in M9 medium, and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher, San Jose, CA, USA) was operated in full scan (120,000 FWHM, 350-1500 m/z) mode at positive ion mode with orbitrap detector. The electro-spray voltage was 2.5 kV, and the heated capillary temperature was 320°C. All the mass spectra were recorded with Xcalibur software (version 3.1, Thermo Fisher Scientific, USA). MS/MS spectra were acquired by data-dependent acquisition mode, and total cycle time was set to 3 s. Peptides with charge state ≥2 were selected for sequencing with HCD (collision energy 32%, max injection time 35 ms) and neutral-loss-triggered (Δ97.98) EThcD (ETD reaction time 50 ms, max ETD reagent injection time 200 ms, supplemental activation energy 25%, max injection time 35 ms) for fragmentation. All product ions were detected in the ion trap (rapid mode).
Database search. Proteome Discoverer 2.1 (for analysis of the phosphorylated peptides in E. coli cultured in LB medium) with MASCOT 2.4 and Maxquant 1.6.0 (for the phosphorylated peptides in E. coli cultured in M9 medium and HeLa) were applied for database searching against the NCBI_E.coli_k12 database (updated on 01/18/2016, 4127 proteins) or Uniprot_Homo Sapiens database (updated on 04/04/2019, 42432 proteins). The corresponding reversed database was also performed to evaluate the false discovery rate (FDR) of peptide identification in the database searching process. The parameters of database searching included: up to three missed cleavages allowed for full tryptic digestion, precursor ion mass tolerance 10 p.p.m., product ion mass tolerance 20 m.m. u., carbamidomethylation (C) as a fixed modification, and oxidation (M), phosphorylation (STYHRK), acetyl (protein N-term), deamidated (NQ) as variable modifications. PSMs were validated using perculator based on q-value at a 1% FDR. GO analysis was carried out on http://pantherdb.org/ (Panther 15.0) and http://revigo.irb.hr/. Sequence motif analysis was carried out on Weblogo 2.8.2.
ITC experiment. ITC titration was performed on an Isothermal Titration Calorimeter from MicroCal Inc. All measurements were conducted at 298 K. In general, a solution of the peptide (1-2 mM) in 50 mM HEPES buffer (pH 7.2) was injected stepwise (10 μL × 24 times) into a solution of DpaZn (25-100 μM) dissolved in the same solvent system. The measured heat flow was recorded as function of time and converted into enthalpies (ΔH) by integration of the appropriate reaction peaks. The binding parameters (K D , ΔH, ΔS, n) were evaluated by applying one site model using the software Origin (MicroCal Inc.).
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