Magnetic mesoporous silica nanocomposites with binary metal oxides core-shell

structure for the selective enrichment of endogenous phosphopeptides from

human saliva

Yilin Lia, Liangliang Liua, Hao Wub*, Chunhui Denga,c*

aDepartment of Chemistry, The Fifth People’s Hospital of Shanghai, Fudan

University, Shanghai 200433, China

b Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan

University, Shanghai 20032, China

c Institutes of Biomedical Sciences, and Collaborative Innovation Center of Genetics

and Development, Fudan University, Shanghai 200433, China

Corresponding Authors

E-mail: chdeng@fudan.edu.cn. (Chunhui Deng)

E-mail: Wu.han@zs-hospital.sh.cn (Hao Wu)

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1. Experimental section

1.1 Material characterization.

Transmission electron microscopy (TEM) images were obtained on a JEOL 2011

microscope (Japan) at 200 kV. Fourier transform infrared spectra (FT-IR) was

operated on a Nicolet Fourier spectrophotometer (U.S.A.). X-ray energy dispersive

spectrometer (EDS) was measured on the field emission transmission electron

microscope. Nitrogen sorption isotherms was measured by Micromeritcs Tristar 3000

analyzer. The Brunauer-Emmett-Teller (BET) surface area was measured on a relative

pressure range from 0.047 to 0.29. BJH (Barrett-Joyner-Halenda) method was

applyed to calculate the pore diameter and distribution curves.

1.2 MALDI-TOF MS analysis.

All MALDI-TOF-MS experiments were carried out in a reflector positive mode by

an AB Sciex 5800 MALDI-TOF mass spectrometer (AB Sciex, USA) with a Nd-YAG

laser at 355 nm, an acceleration voltage of 20 kV and a frequency of 200 Hz, and. The

matrix solution of 2, 5-dihydroxybenzoic acid (DHB, 10 mg mL -1) was dissolved in

ACN/H2O/TFA (50:50:0.1, v/v/v). The eluant (1 μL) was analyzed on the MALDI

plate with 1 μL matrix solution for mass analysis.

1.3 Nano-LC-MS/MS analysis.

Firstly, the lyophilized peptides were resuspended with 10 μL solvent A (water with

0.1% formic acid). Nano-LC was then utilized to separate peptides for on-line

electrospray tandem mass spectrometry analysis. The experiments were performed on

an EASY-nLC 1000 system (Thermo Fisher Scientific, Waltham, MA) connected to

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an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA)

equipped with an online nano-electrospray ion source. 4μl peptide sample was loaded

onto the trap column (Thermo Scientific Acclaim PepMap C18, 100μm x 2cm), with a

flow of 10μl min-1 for 3 min and subsequently separated on the analytical column

(Acclaim PepMap C18, 75μm x 25cm) with a linear gradient, from 2% B to 35% B in

105 min. The column was re-equilibrated at initial conditions for 15 min. The column

flow rate was kept at 300nL/min and column temperature was kept at 40 . The℃

electrospray voltage of 2.0 kV versus the inlet of the mass spectrometer was applied.

The Orbitrap Fusion mass spectrometer was performed in the data-dependent mode to

switch automatically between MS and MS/MS acquisition.

Survey full-scan MS spectra (m/z 350- 1500) were acquired in Orbitrap with a

mass resolution of 120 000 at m/z 200. The AGC taget was set to 1 000 000, and the

maximum injection time was set as 50 ms. MS/MS acquisition was performed in

Orbitrap in 3 s cycle time. Ions with charge states 2+, 3+, and 4+ were orderly

fragmented by HCD with NCE of 35% and fixed first mass was set at 110. The

intensity threshold was 50 000, and the maximum injection time was 100 ms. The

AGC target was set to 200 000, and the isolation window was 2 m z -1. One microscan

was operated with dynamic exclusion of 30 seconds.

1.4 Database Search.

Tandem mass spectra were extracted by Proteome Discoverer software (Thermo

Fisher Scientific, version 1.4.0.288) without charge state deconvolution and

deisotoping. All MS/MS data were analyzed by Mascot (Matrix Science, London,

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UK; version 2.3.2) and the database was the Human UniProtKB/Swiss-Prot database

(Release 2015-03-11, with 20199 sequences). Raw files from the LTQ Orbitrap were

searched with a 10 ppm precursor mass tolerance and a 50 mmu fragment mass

tolerance. The enzyme specificity with trypsin was used. Carbamidomethyl on

cysteine was regarded as a fixed modification. In the meantime, Oxidation on

methionine and deamidation on asparagine were regarded as variable modifications.

Peptide level false discovery rates (FDR) was controlled by the percolator algorithm

to lower than 1%. The identified deamination sites that were confirmed to the N-

glycosylation consensus sequence (n-!P-[S/T]) were regarded as glycosylation sites.

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Element

Peak Area k Abs Weight%

Weight%

Atomic%

Area Sigma

factor Corrn.

Sigma

O K 579 54 1.810

1.000 39.49 2.45 65.47

Si K 307 32 1.000

1.000 11.57 1.19 10.93

Ti K 116 21 1.050

1.000 4.57 0.83 2.53

Fe K 1006

51 1.170

1.000 44.36 2.15 21.07

Totals 100.00

Element

Peak Area k Abs Weight%

Weight%

Atomic%

Area Sigma

factor Corrn.

Sigma

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O K 435 41 1.810 1.000 20.48 1.73 47.81Si K 355 38 1.000 1.000 9.22 0.97 12.26Fe K 141

462 1.170 1.000 43.00 1.94 28.75

Zr K 400 45 2.624 1.000 27.29 2.35 11.17

Totals 100.00Fig. S1. TEM images and elemental analysis of (A) Fe3O4@TiO2@mSiO2 (B)

Fe3O4@ZrO2@mSiO2.

Fig. S2. Fe3O4@TiO2-ZrO2@mSiO2 dispersed in water (A) and separated

magnetically (B).

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Fig. S3. Image of size distribution report of (A) Fe3O4, (B) Fe3O4@TiO2-ZrO2, (C)

Fe3O4@TiO2-ZrO2@mSiO2 by intensity.

Fig. S4. The adsorption-desorption isotherms (A) and pore distribution (B) of

Fe3O4@TiO2-ZrO2@mSiO2.

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Fig. S5. The adsorption-desorption isotherms (A) and pore distribution (B) of

Fe3O4@TiO2-ZrO2.

Table S1. Detailed information of phosphopeptides enriched from β-casein digests by

using Fe3O4@ZrO2@mSiO2, Fe3O4@TiO2@mSiO2 and Fe3O4@TiO2-ZrO2@mSiO2.

[pS] represents for phosphorylated site.PeakNo.

Theoreticalm/z

Peptide Sequence Fe3O4@ZrO2@mSiO2

Fe3O4@TiO2@mSiO2

Fe3O4@TiO2-ZrO2@mSiO2

1 1466.3 TVDME[pS]TEVFTK √ √ √2 1561.8 RELEELNVPGEIVESL[pS]

[pS][pS]EE[pS]ITR√

3 1660.5 VPQLEIVPN[pS]AEER √4 1951.5 YKVPQLEIVPN[pS]AEER √ √ √5 2061.4 FQ[pS]EEQQQTEDELQDK √ √ √6 2431.5 IEKFQ[pS]EEQQQTEDELQ

DK√

7 2556.6 FQ[pS]EEQQQTEDELQDKIHPF

√ √ √

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8 3122.6 RELEELNVPGEIVE[pS]L[pS][pS][pS]EESITR

√ √ √

Fig. S6. Mass spectra of tryptic digested β-casein (1 fmol/μL) obtained: (A) before

enrichment and (B−D) after treatment with (B) Fe3O4@TiO2@mSiO2, (C)

Fe3O4@ZrO2@mSiO2, and (D) Fe3O4@TiO2-ZrO2@mSiO2. Mass spectrometric peaks

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of mono-phosphopeptides were labeled with red square, multi-phosphorylated

peptides with triangles.

Fig. S7. MALDI-TOF mass spectra of tryptic digested β-casein (0.2 fmol/μL) after

enriched by Fe3O4@TiO2-ZrO2@mSiO2. Mass spectrometric peaks of mono-

phosphorylated peptides were marked with red square.

Table S2 The comparison table of Fe3O4@TiO2-ZrO2@mSiO2 and other published

works.

* ACS Appl. Mater. Interfaces. 2014, 6, 11799−11804

** J. Proteome. Res. 2008, 7, 2526–2538

*** J. Proteome. Res. 2007, 6, 4498-4510

**** RSC Adv. 2016, 6, 96210–96222

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Fig S8. MALDI-TOF mass spectra of tryptic digested β-casein (100 fmol/μL) after

treatment with (a) the first-time Fe3O4@TiO2-ZrO2@mSiO2 and (b) the five-times-

recycled Fe3O4@TiO2-ZrO2@mSiO2. Mass spectrometric peaks of mono-

phosphorylated peptides were marked with red square, multi-phosphorylated peptides

with triangles.

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Fig S9. Mass spectra of endogenous phosphopeptides captured from human saliva:

(A) after enriched by Fe3O4@TiO2@mSiO2 and (B) after enriched by

Fe3O4@ZrO2@mSiO2. Mass spectrometric peaks of mono-phosphopeptides were

labeled with red square, multi-phosphorylated peptides with triangles.

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Fig S10. MALDI-TOF MS/MS spectra of endogenous phosphorylated peptides in

human saliva after enriched by Fe3O4@TiO2-ZrO2@mSiO2. Other peaks of

phosphorylated peptides had been proved in former reports [1-3].

Table S3. Detailed information of the identified endogenous phosphopeptides

enriched from non-digested human saliva by Fe3O4@TiO2-ZrO2@mSiO2, given by

nano LC-ESI-MS/MS.

Sequence Number of phosphorylated sites

M

H+

[Da]

DVPLVISDGGDsEQFIDEER 1 2299.99

DGGDsEQFIDEER 1 1576.58

GGDsEQFIDEER 1 1461.55

SDGGDsEQFIDEER 1 1663.61

GGDsEQFIDEERQGPPLGGQQ 1 2323.98

DGGDsEQFIDEERQGPPLGGQQ 1 2439.01

DVPLVISDGGDsE 1 1382.57

sEQFIDEER 1 1232.48

GGDsEQFIDEERQGPPLGGQQSQPS 1 2723.16

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GGDsEQFIDEERQ 1 1589.61

QNLNEDVsQEESPSLIAGNPQGPSPQ 1 2815.24

QNLNEDVsQEESPSLIAGNPQGPSPQGGNKPQ 1 3396.53

QNLNEDVsQEESPSLIAGNPQGPSPQGGNKPQGPPPPPGKPQ 1 4349.04

QNLnEDVsQEESPSLIAGNPQGPSPQGGNKPQ 1 3397.53

QnLNEDVsQEESPSLIAGNPQGPSPQGGnKPQGPPPPPGKPQ 1 4351.05

QNLnEDVsQEESPSLIAGNPQGPSPQGGNKPQGPPPPPGKPQ 1 4350.06

QNLNEDVsQEESPSLIAGNPQGP 1 2503.10

QNLNEDVsQEESPSLIAGnPQGAPPQGGNKPQGPPSPPGKPQ 1 4324.04

QGPPPQGDKsRsPRSPPGKPQGPPPQGGNQPQGPPPPPGKPQ 2 4368.08

SsEEKFLR 1 1075.48

VISDGGDsEQFIDEER 1 1875.76

QDLDEDVSQEDVPLVISDGGDsEQFIDEER 1 3458.48

GGDsEQFIDEERQGPPLGGQQ 1 2323.98

QNLNEDVsQEESPSLIAGnPQGPSPQGGnKPQGPPPPPGKPQ 1 4351.06

DVsQEESPSLIAGNPQGP 1 1904.83

QNLNEDVSQEESPsLIAGNPQGPSPQG 1 2872.27

QNLNEDVsQEESPSLIAGNPQGAPPQGGNKPQ 1 3380.54QNLNEDVsQEESPSLIAGNPQGAPPQGGNKPQGPPSPPGKPQ 1 4323.03

DVsQEESLFLISGKPEGRRPQGGNQPQ 1 3033.44

ssEEKFLR 2 1155.45

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