Smart Hydrophilic Modification of Magnetic Mesoporous Silica with

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Smart Hydrophilic Modification of Magnetic Mesoporous Silica with Zwitterionic L-Cysteine for Endogenous Glycopeptides Recognition Hemei Chen, Yilin Li, Hao Wu, Nianrong Sun, and Chun-hui Deng ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06258 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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ACS Sustainable Chemistry & Engineering

Smart Hydrophilic Modification of Magnetic Mesoporous Silica with Zwitterionic L-Cysteine for Endogenous Glycopeptides Recognition Hemei Chen, 1 Yilin Li, 1 Hao Wu, 2 Nianrong Sun,*, 1 Chunhui Deng*, 1 1

Department of Chemistry, The Fifth People’s Hospital of Shanghai, Institutes of Biomedical Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, 220 Handan Road, Yangpu District, Shanghai 200433, China

2

Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

Corresponding Authors *Dr. N. R. Sun, E-mail: [email protected] *Prof. C. H. Deng, E-mail: [email protected]

1

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Abstract: There are still few reports about endogenous glycosylated peptides although the significance of the study for them has been confirmed since they may provide higher sensitivity and specificity in clinical practice. In this work, magnetic mesoporous silica microspheres were endowed with strong hydrophilicity by a simple and smart modification way to L-Cysteine, which was attributed to the bond formation of Fe-S. The microsphere (denoted as L-Cys-Fe3O4@mSiO2) was composed of Fe3O4 core, mesoporous silica layer and innumerable perpendicularly aligned mesopores, as well as abundant L-Cysteine in the mesopores. Thanks to the excellent hydrophilicity of L-Cysteine probe, the L-Cys-Fe3O4@mSiO2 microspheres showed strong recognition ability towards glycopeptides. And owing to the superparamagnetic cores well encapsulated in microspheres, the whole process is quite time-saving by magnetic separation. Also, due to the appropriate pore size of the L-Cys-Fe3O4@mSiO2 microspheres, the extraction method exhibited good size-exclusion effect towards large-sized proteins. Finally, the glycopeptidome analysis method based on the L-Cys-Fe3O4@mSiO2 microspheres was proved to be highly efficient in enriching endogenous glycopeptides in healthy and gastric cancer human saliva directly. Keywords: Endogenous glycopeptides / Recognition / Mesoporous microspheres / Hydrophilicity

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Protein glycosylation, as one of the most significant post-translational modifications, carries abundant biological information and plays specific roles in many biological events including signal transduction, tumor immunology, cell growth and differentiation, etc.1-4 A great many reports have put forward powerful evidence that many clinical biomarkers and therapeutic targets are glycosylated proteins or peptides.5 Peptidome plays a crucial role in various physiological and pathological processes.6 So far, some endogenous peptides have been found to be closely associated with clinic diseases, and they could provide higher sensitivity and specificity in clinical practice, so they have great potential to become new biomarkers in early medical diagnosis.7,8 Therefore, compared to common peptidomics research and glycoproteomics research, endogenous glycosylated peptides could contain more cancer-specific diagnostic informations. Despite

the

potential

significance

of

glycopeptidomics, there

are

still

few reports on the post-translational modifications of peptidome, especially on the endogenous glycosylated peptides, owing to some serious barriers in glycopeptidome research. First of all, the high dynamic range of biologic sample makes glycopeptidome analysis a great challenging task.9 Secondly, similar to the peptidome, traditional sample preparation techniques in proteomics, such as centrifugal ultrafiltration with accurate MW cutoff, cannot be applied to endogenous glycosylated peptides owing to their low molecular weight (mostly lower than 20 kDa). Thirdly, compared with non-glycosylated peptides, endogenous glycopeptides usually account for only 2– 5% of total endogenous peptides.10 What’s more, the ionization efficiency of glycopeptides is poor due to the presence of glycan.11 Although mass spectrometry (MS) has been developed into a convenient, high-throughput and efficient technique platform for proteomics and peptidomics research because of its remarkable detection capability with fast speed,11,12 it is hard to direct 3



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analyze endogenous glycopeptides because of the particularity of glycopeptidomics. Therefore, the complex samples must be separated efficiently by a specific method to decrease the complexity of sample matrices before MS analysis. For peptidome research, many researchers focus on the mesoporous material, a representative porous material. Owing to its remarkable features such as uniform pore channels, adjustable pore size, large surface area, modifiable in-pore surface and so on,13,14 functionalized mesoporous material could serve as a recognition platform for endogenous peptides. To date, different kinds of functionalized mesoporous materials, including mesoporous silica, and mesoporous titania etc., have been used to selectively enrich peptides, but exclude high-MW proteins based on size-exclusion mechanism.15,16 In addition, the application of magnetic nanomaterials to proteome research have attracted the general attention, because it is easy to isolate the magnetic materials from the solution by magnetic separation.17,18 In our previous work, magnetic mesoporous silica microspheres with highly ordered periodic mesostructure and mesoporous cupric ion modified magnetic microspheres were developed and applied for peptidomics research.19,20 Also, some strategies have been applied in glycopeptides enrichment, such as lectin affinity, boronate affinity, hydrazide chemistry, metal coordination, etc.21-23 In recent years, by taking advantage of the hydrophilicity difference between glycopeptides and non-glycopeptides, hydrophilic interaction chromatography (HILIC) approach is widely used in separating the glycopeptides, and HILIC exhibits higher glycosylation coverage, unbiased recognition ability for glycopeptides, and excellent good MS compatibility.24-26 Compared with the common HILIC stationary phases,27-30 zwitterionic HILIC (ZIC-HILIC) materials show better hydrophilicity, because they have both positive and negative groups.31-35 4


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Considering the excellent enrichment performance of ZIC-HILIC probe for glycosylated peptides and the merit of mesoporous materials in peptidomics research, we fabricated, for the first

time,

L-Cys

modified

magnetic

mesoporous

silica

materials

(denoted

as

L-Cys-Fe3O4@mSiO2) by a facile Fe–S interaction route for highly specific recognition of endogenous N-linked glycopeptides.33 Owing to the magnetite cores, the specific exclusion for macromolecular protein, and the outstanding affinity for glycopeptides, they hold great promise for fast magnetic separation and high absorption of trace endogenous glycopeptides in liquid systems. In this study, we also have found that L-Cys-Fe3O4@mSiO2 microspheres possess some remarkable features that are useful in endogenous glycopeptides recognition.

Scheme 1. Schematic diagram of the preparation of L-Cys-Fe3O4@mSiO2 microspheres.

EXPERIMENTAL SECTION Materials and Chemicals. Tetraethyl orthosilicate (TEOS), Cetyltrimethylammonium bromide (CTAB), Ammonium bicarbonate (NH4HCO3), horseradish peroxidase (HRP), immunoglobulin G 5



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(IgG), bovine serum albumin (BSA), ovalbumin (OVA) and 2, 5-dihydroxy-benzoic acid (DHB) were purchased from Sigma-Aldrich (USA). L-Cysteine (L-Cys) was purchased from Adamas-beta. PNGase F was purchased from Genetimes Technology. Acetonitrile (ACN) of chromatographically pure was purchased from Merck (Darmstadt, Germany). All deionized water in the experiment was acquired by Milli-Q system (Millipore, Bedford, MA). All of other chemicals are analytically pure. Saliva samples were provided by healthy woman. Synthesis

of

L-Cys-Fe3O4@mSiO2

microspheres.

The

procedures

for

synthesis

of

L-Cys-Fe3O4@mSiO2 microspheres are displayed in Scheme 1. Firstly, the Fe3O4 magnetic nanoparticles were prepared by a solvothermal reation according to our previous method. Then the Fe3O4 particles were coated with mesoporous silica shell (ca. 70 nm in thickness). Briefly, 500 mg CTAB was dissolve in 50 mL deionized water, and then 50 mg Fe3O4 nanoparticles were added into the mixed solution. After ultrasonication for 30 min, 50 mL of 10 mM NaOH and 400 mL of deionized water were added into the mixture and mechanically stirred at 60 °C for 30 min. Then 2.5 mL of TEOS/ethanol (v/v = 1:4) were added into the above solution. The resultant solution was mechanically stirred and heated at 60 °C for 12 h. Following that, the materials were separated with magnet and washed with deionized water and ethanol three times sequentially, and then dried in vacuum at 50 °C over 12 h, followed by calcining at 350 °C for 4 h to remove CTAB. Finally, 15 mg mesoporous silica coated Fe3O4 (denoted as Fe3O4@mSiO2) was redispersed into the mixture containing 15 mg L-Cys and 25 mL PBS buffer (0.01 M), then the mixed solution was stirred at 25 °C for 12 h. The obtained precipitate (L-Cys-Fe3O4@mSiO2) was washed with deionized water for six times and dried in vacuum at 50 °C. Sample preparation. Standard protein (HRP or BSA) was dissolved in 25 mM NH4HCO3 6


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(pH=7.9) and denatured at 95 °C for 10 min. Once cooling down to room temperature, the solution was treated with trypsin (protein/Trypsin=40/1, w/w) at 37 °C for 16 h. Human saliva samples were taken from a young female healthy volunteer and an elderly male gastric cancer volunteer. An equal volume of saliva sample was mixed with 0.2% TFA solution. After high speed centrifugation, deposit of which was removed. The final saliva sample was stored at -80 °C for the following experiments. Characterization and Measurement. The detailed information of characterization and measurement was shown in the Supporting Information. Protocol of glycopeptides enrichment from biological samples. 100 μg L-Cys-Fe3O4@mSiO2 microspheres were added into 100 μL loading buffer (ACN/H2O/TFA=85/15/0.1, v/v/v) with 100 fmol/μL HRP digests. The The obtained solution was incubated at 1000 rpm at 37 °C for 30 min and then the L-Cys-Fe3O4@mSiO2 microspheres with captured peptides were separated by magnet, followed by washing with loading buffer (100 μL) three times to remove non-glycosylated peptides. The obtained glycopeptides were eluted with 10 μL ACN/H2O/TFA (30/70/0.1, v/v/v) at 37 °C for 30 min. Finally, the eluted peptides were directly transferred onto plate for MALDI-TOF MS Analysis. For enrichment of endogenous glycopeptides from saliva samples, 10 μL sample as prepared above (containing 5 μL original saliva sample) was mixed with 90 μL loading buffer and incubated with 500 μg L-Cys-Fe3O4@mSiO2 microspheres at 4 °C for 30 min and subsequently washed with loading buffer (200 μL) four times. The captured glycopeptides were eluted by 40 μL ACN/H2O/TFA (30/70/0.1, v/v/v) at 37 °C for 30 min and then the elution was lyophilized. Following this, the lyophilized glycopeptides was redissolved in 50 μL NH4HCO3 solution (25 7



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mM), and PNGase F was added with the PNGase F/glycopeptides ratio of 100 U/1 μg. After incubation at 37 °C for 16 h to remove N-linked glycans, the solution was lyophilized and redissolved prior to UPLC-MS/MS analysis. MS analysis and Database Search. The detailed information about MS analysis and Database Search were shown in the Supporting Information. RESULTS AND DISCUSSION Synthesis and Characterization of Fe3O4@mSiO2 microspheres. The synthetic procedure of L-Cys-Fe3O4@mSiO2 microspheres was illustrated in Scheme 1. Briefly, Fe3O4 particles were firstly synthesized by the hydrolysis. Then the perpendicularly aligned mesoporous SiO2 shell was introduced onto the surface of the Fe3O4 particles via a surfactant-assembly sol-gel process. After being calcined to remove the surfactant of CTAB, the Fe3O4@mSiO2 microspheres were mixture with L-Cys in PBS buffer to complete the functionalization. Thus it can be seen the whole synthetic process was fairly facile and timesaving compared with the previous report.36

8


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Figure 1. TEM images of: (a), (b) Fe3O4@mSiO2 microspheres; (c) Nitrogen adsorption-desorption isotherms of L-Cys-Fe3O4@mSiO2 (The pore size distribution profile was inserted).

The transmission electron microscopy (TEM) was used to characterize the morphology of Fe3O4@mSiO2 microspheres. It showed that the Fe3O4 core was about 250 nm in diameter (Figure 1a), and it was well encapsulated in mesoporous SiO2 shell, an ordered mesoporous silica phase (~ 20 nm in thickness) with cylindrical channels (Figure 1a, b). The Nitrogen adsorption-desorption 9



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isotherm measurements of Fe3O4@mSiO2 microspheres (Figure S1 in the Supporting Information) and L-Cys-Fe3O4@mSiO2 microspheres (Figure 1c) suggested representative type-IV due to the cylindrical mesoporous channels. The BET surface area and total pore volume of Fe3O4@mSiO2 microspheres were estimated to be 224.6 m2/g and 0.21 cm3/g respectively. The pore size distribution curve (Figure S1, insert) showed the pore size was around 3.2 nm. After modified with L-Cys, The BET surface area of L-Cys-Fe3O4@mSiO2 microspheres was calculated to be 194 m2/g and the total pore volume was about 0.17 cm3/g. The calculated pore-size distribution indicated that the average pore size of the functional microspheres was still about 3.2 nm that mainly due to the immobilization of L-Cys on the surface of Fe3O4 through the mesoporous channels instead of immobilization on the mesoporous inwall (Figure 1c, insert). However, after modification with L-Cys, the microspheres decreased in surface area and total pore volume, indicating the successful binding of L-Cys to some extent.

10


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Figure 2. FT-IR spectra of (a) Fe3O4, (b) Fe3O4@mSiO2, (c) L-Cys-Fe3O4@mSiO2, (d) L-Cysteine

11



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Figure 3. The XPS spectra of L-Cys-Fe3O4@mSiO2 microspheres.

FT-IR

spectroscopy

was

further

employed

to

characterize

L-Cys-Fe3O4@mSiO2

microspheres (Figure 2). A strong absorption peak at about 563 cm−1 which can be assigned to the characteristic peak of Fe–O was all clear in the spectra of Fe3O4, Fe3O4@mSiO2 and L-Cys-Fe3O4@mSiO2. The peak at about 1084 cm-1 in the spectra of Fe3O4@mSiO2 and L-Cys-Fe3O4@mSiO2 were attributed to the asymmetric stretching vibration of Si−O−Si, indicating the well synthesis of Fe3O4@mSiO2. Compared with the spectra of Fe3O4@mSiO2, many significant fingerprint peaks (from 1400 to 400, marked with a dotted box) appeared in the spectra of L-Cys-Fe3O4@mSiO2, which were exhibited in the spectra of pure L-Cys, suggesting the successful fabrication of L-Cys-Fe3O4@mSiO2 microspheres. In the energy dispersive X-ray (EDX) analysis of L-Cys-Fe3O4@mSiO2 microspheres, the appearance of S element confirmed the well modification of L-Cys on the microspheres further (Figure S2 in the Supporting Information). 12


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Additionally, X-ray photoelectron spectroscopy (XPS) was further employed to characterize the element composition and bonding mode of L-Cys-Fe3O4@mSiO2 microspheres. As shown in Figure 3, evident peaks of Fe 2p, C 1s, O 1s, N 1s, Si 2s, Si 2p3 and S 2p3 were observed, indicating the presence of L-Cys, SiO2 and Fe3O4. Then the Fe 2p3/2-1/2 peak was studied by peak-differenating analysis, and it showed that the peak fitting were made up of Fe-O bond and Fe-S bond (Figure S3), which could directly prove the formation of Fe-S bond.23,37,38

Figure 4. TGA curves of Fe3O4@mSiO2 and L-Cys-Fe3O4@mSiO2 microspheres

In the meantime, thermogravimetric analysis (TGA) was conducted to evaluate the amount of immobilized L-Cys. As shown in Figure 4, L-Cys-Fe3O4@mSiO2 microspheres displayed a larger mass loss than Fe3O4@mSiO2 with the increase of temperature, which was considered as the loss of L-Cys. From TGA data at 800 °C, the amount of L-Cys on L-Cys-Fe3O4@mSiO2 microspheres was about 7.4%, which was larger than other reported mesoporous silica materials,25,26 implying a better hydrophilicity. The superior hydrophilicity was furtherly demonstrated by measuring the 13



Page 14 of 25

water contact angle of L-Cys-Fe3O4@mSiO2 microspheres. As shown in Figure S4, L-Cys-Fe3O4@mSiO2 microspheres exhibited 15.14° of water contact angle, which was smaller than previous report.39 Moreover, the water contact angle of Fe3O4@mSiO2 microspheres was also measured to be about 20.02°, which was bigger than that of L-Cys-Fe3O4@mSiO2 microspheres, indicating the importance of L-Cys for improving hydrophilicity. The first advantage of this enrichment process is that the isolation speed was fast without reiterative centrifugation compare with that of nonmagnetic materials methods.31 As shown in Figure S5a, L-Cys-Fe3O4@mSiO2 microspheres had high magnetization of about 50.0 emu/g in saturation value. They could be well dispersed in water and rapidly realized solid-liquid separation by external magnet in five seconds (Figure S5b, c), which suggested the super magnetic response of L-Cys-Fe3O4@mSiO2 microspheres.

Scheme 2. Flowchart of the enrichment process with L-Cys-Fe3O4@mSiO2 microspheres.

Optimization of enrichment conditions of L-Cys-Fe3O4@mSiO2 microspheres. The enrichment procedure for glycopeptides from complex samples by using L-Cys-Fe3O4@mSiO2 microspheres was displayed in Scheme 2. To optimize the extraction of peptides from sample solutions, the factors that potentially affected the interactions of L-Cys-Fe3O4@mSiO2 14


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microspheres and glycopeptides, such as loading buffer, incubation time, eluting solution, eluting time and material amount were studied with HRP digest solution through five parallel experiments. Five different loading buffers were examined for the purpose of obtaining better results. By comparing the number of matched glycopeptides and peak intensity (the strongest) of the experiments, 85% ACN/0.1% TFA (v/v) aqueous solution was chosen as optimized loading buffer (Figure S6a). Six different incubation times ranging from 5 to 60 min were investigated for the extraction of glycopeptides from tryptic digests of HRP. From Figure S6b, there is no significant difference in the enrichment performance from 10 to 30 min, suggesting the rapid affinity of L-Cys-Fe3O4@mSiO2 microspheres for glycopeptides. Moreover, it can be seen that the matched peptides or the peak intensity became a bit lower when the incubation time was longer than 30 min, this should mainly laid the blame at the increasing nonspecific adsorption along with the increase of incubation. In the same way, to obtain the full elution of captured glycopeptides, four different eluting solutions and six different elution time ranging from 5 to 60 min were examined. According to Figure S6c, 30% ACN/0.1% FA (v/v) aqueous solution was selected as optimized eluting solution. And from Figure S6d, when the elute time was over 20 min, it did not show significant change in the peak intensity and the matched glycopeptides, suggesting 20 min was enough to elute the glycopeptides from L-Cys-Fe3O4@mSiO2 microspheres. The slight change shown in Figure S6d may be caused by the systematic error of detection apparatus and manual operation. At last, according to Figure S6e, 100 μg was the optimal material amount for 100 μL HRP digests (100 fmol/μL). When the amount of L-Cys-Fe3O4@mSiO2 microspheres was larger than 150 μg, the decrease of intensity and the matched number may be caused by the incomplete elution under the condition of the same amount of eluent but excess material amount. 15



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Thus, with magnetic separation, an effective zwitterionic-HILIC approach for glycopeptide recognition was established. Investigation of common performance of L-Cys-Fe3O4@mSiO2 microspheres. HRP digest solution was chosen as standard sample to evaluate the sensitivity of L-Cys-Fe3O4@mSiO2 microspheres in glycopeptide enrichment. As shown in Figure 5a, only three glycopeptides with low signal intensity could be observed for original HRP digest solution. However, after enrichment with the L-Cys-Fe3O4@mSiO2 microspheres, 21 glycopeptides with high peak intensity were assigned (Figure 5b) and their detailed information is shown in Table S1 (Supporting Information). Following this, HRP digests with lower concentration were employed in the enrichment process. 13 glycopeptide peaks could be obtained when the HRP concentration was 10 fmol/μL (Figure 5c) and 4 glycopeptide peaks could be gotten even at the ultralow concentration of 1 fmol/μL after treatment with L-Cys-Fe3O4@mSiO2 microspheres (Figure 5d). In addition, another protein digest (IgG) which contains different glycopeptides was used to investigate

the

common

performance

of

L-Cys-Fe3O4@mSiO2

microspheres

towards

glycopeptides (Figure S7). Before treatment, there was no glycopeptides could be observed for 100 fmol/μL IgG digest solution. However, after enrichment with the L-Cys-Fe3O4@mSiO2 microspheres, 26 glycopeptides with high peak intensity were assigned (Figure S7b, Table S2). The

result

sufficiently

proved

the

remarkable

glycopeptides

enriching

capacity

of

L-Cys-Fe3O4@mSiO2 microspheres.

16


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Figure 5. MALDI-TOF MS spectra of: (a) 100 fmol/μL HRP tryptic digests direct analysis, and 100 μL HRP tryptic digests with concentration of: (b) 100 fmol/μL, (c) 10 fmol/μL, (d) 1 fmol/μL after enrichment with L-Cys-Fe3O4@mSiO2 microspheres. The peaks of glycopeptides were marked in red circles.

Also, a more complex solution, mixture of HRP tryptic digest and tryptic digest of non-glycoprotein BSA with different mass ratios, were used to further examine the recognition capacity of L-Cys-Fe3O4@mSiO2 microspheres towards glycopeptides. As shown in Figure S8a, glycopeptides could be hardly identified with mass ratio at 1:20 (HRP:BSA) owing to the disturbance

of

non-glycopeptides

from

BSA

tryptic

digest.

After

treatment

by

L-Cys-Fe3O4@mSiO2 microspheres, the signals of 15 glycopeptides were assigned (Figure S8b). Even when the mass ratio changed to 1:100, the result still reflected L-Cys-Fe3O4@mSiO2 microspheres possessed good selective recognition ability towards glycopeptides (Figure S8c), indicating that they could be applied for glycopeptidome study.

17



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Figure 6. MALDI mass spectra of HRP digests after enrichment with the same L-Cys-Fe3O4@mSiO2 microspheres (a) for the first time and (b) for the fifth time. The peaks of glycopeptides were marked in red circles.

The

second

advantage

of

the

enrichment

process

is

that

the

reusability

of

L-Cys-Fe3O4@mSiO2 microspheres was remarkable owing to their stability. L-Cys-Fe3O4@mSiO2 microspheres were alternately washed with loading buffer and eluting solution for several times to remove contaminants before next experiment. As shown in Figure 6, there was no significant difference in the enrichment performance from the first and the fifth result, suggesting the super stability of as-synthesized L-Cys-Fe3O4@mSiO2 microspheres in repeated enrichment of glycopeptides.

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Figure 7. MALDI-TOF MS spectra of the mixture of HRP tryptic digests and OVA protein with a mass ratio of 1:20 before treatment (a, b), and 1:20 (c, d), 1:100 (e, f), 1:500 (g, h) after treatment with L-Cys-Fe3O4@mSiO2 microspheres. The peaks of glycopeptides were marked with red circles. Analysis of proteins was performed in the linear TOF detection modes.

Investigation of size-exclusion performance of L-Cys-Fe3O4@mSiO2 microspheres. The third main advantage of this enrichment strategy is that the L-Cys-Fe3O4@mSiO2 microspheres can selectively concentrate glycopeptides but exclude the coexisted large sized proteins simultaneously. The suppression of high abundance proteins are critical issues in the analysis of endogenous peptides, and removing them by mesoporous materials with controlled pore size has been proved to be one of the most ideal methods in peptidome research.14,15 The size-exclusion property of L-Cys-Fe3O4@mSiO2 microspheres was investigated by using the mixture of HRP digest and OVA (a glycoprotein with molecular weight of about 45,000 Da). As shown in Figure 7a and 7b, none of glycopeptides could be detected apart from the clear peaks of OVA protein 19



Page 20 of 25

when the mass ratio of HRP digests/OVA was 1:20 (100 μL containing 0.4 μg HRP digests). While after enrichment with L-Cys-Fe3O4@mSiO2 microspheres, 17 HRP glycopeptide peaks were clearly observed with enhanced signal intensity (Figure 7c), at the same time, the peaks of OVA protein disappeared (Figure 7d). As a comparison, L-Cys-Fe3O4 particles and Fe3O4@mSiO2 microspheres were used to treat the same mixture of HRP digest and OVA (at ratio of 1:20). As displayed in Figure S9a and S9b, after enrichment with L-Cys-Fe3O4 particles, obvious OVA protein peaks and only 10 HRP glycopeptide peaks could be observed, this result indicated the L-Cys-Fe3O4 without mesoporous silica could not show good size-exclusion performance. And as displayed in Figure S9c and S9d, there was no glycopeptide or glycoprotein showed up in mass spectra, indicating the disability of Fe3O4@mSiO2 microspheres for glycopeptides enrichment. However, when the mass ratio was further increased to 1:100 and even 1:500, after treatment with L-Cys-Fe3O4@mSiO2 microspheres, 14 and 9 HRP glycopeptides peaks could still be clearly identified respectively (Figure 7e and 7g). All the above results demonstrated the glycopeptides enrichment ability and size-exclusion ability of L-Cys-Fe3O4@mSiO2 microspheres, while L-Cys-Fe3O4 without mesoporous silica and Fe3O4@mSiO2 could not show good performance. Enrichment of endogenous glycopeptides from human saliva by L-Cys-Fe3O4@mSiO2 microspheres. To further confirm their superior enrichment efficiency for endogenous glycopeptides, L-Cys-Fe3O4@mSiO2 microspheres were applied to endogenous glycosylation analysis in real biological samples. Human saliva, one of non-invasively obtained human specimen of clinical significance, is considered great potential in exploring the biomarkers for diseases or cancers. Here, human saliva samples from healthy volunteer or gastric cancer volunteer were used as practical sample. After the captured endogenous glycopeptides were 20


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eluted, they were further treated with PNGase F to remove N-linked glycans, and then the deglycosylated peptides were further analyzed by UPLC-MS/MS. As shown in Table S3 and Table S4, a total of 46 and 36 endogenous glycopeptides were identified from healthy and gastric cancer volunteer’s saliva. Compared with the reported results,26,36 L-Cys-Fe3O4@mSiO2 microspheres displayed the better sensitivity, suggesting great potential in endogenous glycopeptides identification of biosample (the detailed comparison information was listed in Table S5). CONCLUSIONS In summary, L-Cys-Fe3O4@mSiO2 microspheres were facilely synthesized by combing sol-gel approach in the act of template and seeding thiol reagents via Fe–S interaction. The synthesized L-Cys-Fe3O4@mSiO2 microspheres have magnetite cores, perpendicularly aligned mesoporous SiO2 shell, and enough zwitterionic hydrophilic L-Cys for admirable adsorption. These microspheres was successfully applied to enrich endogenous glycopeptides in human saliva directly, effectively and conveniently, which suggested they were promising hydrophilic probe for the application of exploration of glycopeptidome biomarkers. ASSOCIATED CONTENT Supporting Information Supporting Information Available: [L-Cys-Fe3O4@mSiO2] This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS 21



This

work

was

financially

supported

by

National

Key

R&D

Page 22 of 25

Program

of

China

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TABLE OF CONTENTS (TOC) GRAPHIC:

Synopsis: L-Cysteine modified magnetic mesoporous silica microsphere was synthesized for highly selective recognition of endogenous glycopeptides based on superior hydrophilicity and size-exclusion effect.

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Smart Hydrophilic Modification of Magnetic Mesoporous Silica with

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