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Hydrophilic Mesoporous Silica Materials for Highly Specific Enrichment of N-linked Glycopeptide Nianrong Sun, Jiawen Wang, Jizong Yao, and Chun-hui Deng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04054 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Analytical Chemistry

1

2

Hydrophilic Mesoporous Silica Materials for Highly

3

Specific Enrichment of N-linked Glycopeptide

4

Nianrong Sun, Jiawen Wang, Jizong Yao, Chunhui Deng∗,

5

Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai

6

200433, China.

7

1 ACS Paragon Plus Environment


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ABSTRACT: Hydrophilic interaction liquid chromatography (HILIC) is a significant enrichment

2

strategy in glycoproteomics profiling. In this report, hydrophilic magnetic mesoporous silica

3

materials (denoted as Fe3O4@mSiO2-IDA) were designed and synthesized as an outstanding

4

enrichment platform for glycopeptide analysis. By taking advantage of their merits, such as large

5

surface area, excellent hydrophilicity and unbiased affinity toward all types of glycopeptides, the

6

Fe3O4@mSiO2-IDA nanomaterials were successfully applied to capture glycopeptides from

7

complex samples. A total of 25 glycopeptides from horseradish peroxidase (HRP) digests and 33

8

glycopeptides from Ig G were identified, respectively. Especially, as a result, 424 glycopeptides

9

assigned to 140 glycoproteins were identified from only 2 µL human serum.

10 11


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Glycosylation, one of the most prevalent and significant protein post-translational modification

2

in mammals, due to the crucial roles it plays in regulating numerous biological processes, has

3

been paid much attention.1 Carbohydrate compositions, species and branching structure and so

4

on, those changes in which are closely related to a number of occurrence and development of

5

disease, especially evolution and progression in tumour.

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relevance between glycosylation and disease, it is vital to identify more structural information on

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glycosylation sites, the corresponding glycan structural information and the related peptide

8

information. 4 However, many factors, including the low abundance and ionization efficiency of

9

glycopeptides and the existence of a large number of non-glycopeptides, make the identification

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of glycopeptides encounter great challenge for mass spectrometry (MS)-based strategies. An

11

efficient solution is to conduct an enrichment process prior to MS analysis, which can increase

12

the concentration of the glycopeptides and remove other constituents simultaneously. 4,5

2,3

To get insight into the detailed

13

Efforts have been made in the development of diverse techniques to enrich glycopeptides, and

14

one prominent method is the hydrophilic interaction liquid chromatography (HILIC), which lies

15

in the fact that non-glycopeptides are generally less hydrophilic than glycopeptides.

16

approaches show many advantages with MS analysis, such as excellent reproducibility, high

17

enrichment performance and unbiased enrichment towards various glycopeptides, which make it

18

a promising way to enrich glycopeptides. And many biocompatible nanomaterials, which were

19

synthesized by introducing hydrophilic functional groups onto the surface of the carrier, have

20

been applied to selectively separate and enrich glycopeptides.9 In general, diverse

21

functionalization grafting need different synthetic techniques, and complicated functionalized

22

processes have to be carried out to acquire the functional moieties because of the limitation of

6-8

HILIC



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1

intrinsic properties of various nanomaterials. According to Zhang’s report,

2

mercapto-polyethylene glycol was immobilized onto the surface of magnetic oxidized graphene

3

by using polyether imide as the reducing and stabilizing reagent of Au nanoparticles. This whole

4

synthetic route, as well as some of other synthetic techniques with tedious manipulations and

5

harsh conditions, not only makes the functionalized processes time-consuming but also results in

6

relatively low deposition of hydrophilic groups on the surface of nanomaterials. It has reported

7

that varieties of carries including graphene oxide, 6 metal-organic frameworks, 7 silica

8

magnetic nanoparticles 8,12,13 have been chosen as substrates for functional groups grafting. And

9

the functional groups introduced onto the surface of those carriers included chitosan,14 glucose,15 cyclodextrins,17,18

amine,20

and

poly-saccharide,16

11

glycol,10,21zwitterionic polymers 8 and so forth. And it also has been demonstrated that the more

12

functional groups grafted on the surface of HILIC carriers, the better enrichment performance

13

could be achieved.

14

specific enrichment, that would be valuable for glycoproteomic research.

amino

acid,9

9,11,12

10

21

maltose,13,19

for instance, the

polyethylene

Hence, it is a great demand to develop a facile functionalization route for

15

In virtue of their relatively large surface area, strong stability, uniform mesoporous channels

16

and easily functionalized inwalls, mesoporous silica nanomaterials have exhibited a broad

17

developing prospect for selectively capturing small-size targets from complex samples. In our

18

previous reports, many functionalized mesoporous silica nanomaterials were designed and

19

prepared to enrich endogenous peptides or modified peptides for peptidome research. 22-26

20

In this work, for the first time, we grafted the iminodiacetic acid (IDA) groups facilely onto the

21

inwalls of magnetic mesoporous silica (mSiO2) nanomaterials for highly specific enrichment of

22

N-linked glycopeptides. IDA is a zwitterionic group which has been confirmed to possess

23

excellent hydrophilicity.

27

It was immobilized onto the mesoporous inwalls by using a kind of


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silane coupling agent (3-Glycidoxypropyldimethoxymethylsilane, GLYMO). The most major

2

advantage of the obtained nanomaterials is their wonderful ability to glycopeptides enrichment

3

whether from standard sample by trypsin digest or from practical human serum bio-sample by

4

enzymatic hydrolysis.

5 6

EXPERIMENTAL SECTION

7

Materials and chemicals. Tetraethyl orthosilicate (TEOS), cetyltrimethyl ammonium

8

bromide (CTAB), 3-glycidoxypropyldimethoxymethylsilane (GLYMO) and iminodiacetic acid

9

(IDA) of analytical grade were purchased from Shanghai Chemical Corp. 2, 5-dihydroxy-

10

benzoic acid (DHB), horseradish peroxidase (HRP), Bovine serum albumin (BSA), and

11

immunoglobulin G (IgG) were purchased from sigma aldrich. PNGase F was purchased from

12

Genetimes Technology. Human serum was offered by Shanghai Zhongshan Hospital from a

13

healthy volunteer. Acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). All

14

deionized water in the experiment are prepared by Milli-Q system (Millipore, Bedford, MA). All

15

of other chemicals are of analytical grade.

16

Preparation of GLYMO-IDA solution. Firstly, 2.5 g IDA was dissolved in 50 mL 2 M

17

Na2CO3, and pH was tuned to 10 with 10 M NaOH. The solution was stirred in the condition of

18

ice bath for 10 min, and then 1.6 mL GLYMO was added into the solution drop by drop. After

19

stirring at 0 ℃ for 30 min, the temperature rised to 65 ℃ for another stirring with 6 h. 1.6 mL

20

GLYMO was added into the mixture again after being cool down to 0 ℃. Repeat the above steps

21

and the obtained product was adjusted to pH 6 with concentrated HCl after naturally cooling.

22

And the terminate product was denoted as GLYMO-IDA solution.



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Preparation of Fe3O4@mSiO2-IDA nanomaterials. Firstly, 1.36 g FeCl3•6H2O and 75mL

2

ethylene glycol were blended by magnetic stirring. Then 3.6 g sodium acetate were added into

3

the above mixture. After 30 min, the obtained mixed solution was transferred into a teflon-lined

4

stainless-steel autoclave at 200 ℃ for 12 h. The obtained Fe3O4 particles were washed with

5

deionized water and ethanol for three times in order, and dried in vacuum at 50 ℃ overnight.

6

Secondly, 50 mg Fe3O4 particles were redispersed in a mixed solution containing 500 mg

7

CTAB and 50 mL deionized water for ultrasonication with 30 min. Then 400 mL deionized

8

water and 50 mL 10 mM NaOH were added into the mixture. The mixed solution was stirred at

9

60 ℃ for 30 min before 2.5 mL TEOS/Ethanol(v/v=1:4) were added. The resultant dispersion

10

was continuously heated at 60 ℃ overnight. The obtained particles were washed with deionized

11

water and ethanol for three times in order and dried in vacuum at 50 ℃ overnight. Then the

12

product was treated at 350 ℃ in muffle furnace for 4 h to remove CTAB.

13

Finally, 50 mg Fe3O4@mSiO2 were added into the GLYMO-IDA solution and then the

14

solution was treated ultrasonically for 10 min. The suspension was then heated to 95 ℃ and

15

stirred for 2 h. The product, denoted as Fe3O4@mSiO2-IDA, was washed with deionized water

16

for three times and dried in vacuum at 50 ℃ overnight.

17

Preparation of Fe3O4@nSiO2-IDA nanomaterials. Briefly, Fe3O4 particles were

18

synthesized according to the synthetic procedure of Fe3O4@mSiO2-IDA nanomaterials. Then

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50 mg Fe3O4 particles were dissolved in 450 mL deionized water with ultrasonic dispersion for

20

30 min. Then 50 mL 10 mM NaOH were added into the mixture. The mixed solution was stirred

21

at 60 ℃ for 30 min before 2.5 mL TEOS/Ethanol(v/v=1:4) were added. The resultant dispersion

22

was continuously heated at 60 ℃ overnight. The obtained particles (denoted as Fe3O4@nSiO2) 6 ACS Paragon Plus Environment

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were washed with deionized water and ethanol for three times in order and dried in vacuum at 50

2

℃ overnight. Finally, 50 mg Fe3O4@nSiO2 were added into the GLYMO-IDA solution and and

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then the solution was treated ultrasonically for 10 min. The suspension was then heated to 95 ℃

4

and stirred for 2 h. The product, denoted as Fe3O4@nSiO2-IDA, was washed with deionized

5

water for three times and dried in vacuum at 50 ℃ overnight.

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Tryptic digestion of the standard proteins and human serum. 2 mg HRP (or IgG) and

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500 µL ammonium bicarbonate (50 mM, pH 8.3) were blended by shaking, and then protein

8

solution was denatured by boiling for 10 min. Afterwards, 500 µL Milli Q was added into the

9

solution for making the terminated concentration 2 mg/mL. Also, trypsin (Trypsin/protein=1/40,

10

w/w) was then added into the solution for 16 h at 37 ℃.

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For the preparation of human serum digestion, 2 µL human serum and 16 µL ammonium

12

bicarbonate (25 mM, pH 7.9) were blended homogeneously, and then serum solution was

13

centrifuged at 12 000 rpm for 2 min. The acquired supernatant was reduced by DTT for 30 min

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at 37 ℃, and then alkylated by IAA in the dark situation for 1 h at 37 ℃. 69 µL ammonium

15

bicarbonate (25 mM, pH 7.9) and Trypsin (Trypsin/protein=1/40, w/w) were added for 16 h at 37

16

℃. Tryptic digestion was lyophilized for further use.

17

Protocol of enrichment process. 200 µg Fe3O4@mSiO2-IDA nanomaterials were dispersed

18

in 100 µL loading buffer (ACN/H2O/TFA=89/8/3, v/v/v) containing 1 pmol/µL HRP digestion

19

for being incubated at 37 ℃ with 20 min. After that, the nanomaterials were separated from

20

supernatant

21

(ACN/H2O/H3PO4=85/14.5/0.5, v/v/v) for three times. Then the captured glycopeptides were

with

a

magnet

and

washed

with

200

µL

washing

buffer



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eluted by 6 µL 50% ACN for 30 min at 37 ℃. Finally, the eluent was analyzed directly by

2

MALDI-TOF MS, or lyophilized and deglycosylated for LC-MS/MS analysis.

3

For glycopeptides enrichment from serum, the above-mentioned lyophilized serum was

4

redissolved in 200 µL loading buffer (ACN/H2O/TFA=90/8/2, v/v/v), and then 400 µg

5

Fe3O4@mSiO2-IDA nanomaterials were added to the solution. The mixture was incubated on a

6

platform shaker for 30min at 37 ℃. Then the nanomaterials were separated from supernatant

7

with a magnet and washed with 200 µL washing buffer (ACN/H2O/H3PO4=85/14.5/0.5) for three

8

times. After that, glycopeptides were eluted with 30 µL eluting buffer (50% ACN) twice. Finally,

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the eluted glycopeptides were lyophilized and deglycosylated for LC-MS/MS analysis.

10

Deglycosylation of N-linked glycopeptides by PNGase F. For enriched glycopeptides

11

from serum, 500 units of PNGase F and the solution (ammonium bicarbonate, 60 µL) were

12

blended homogeneously, and then the mixture was disposed at 37 ℃ overnight. In the end, the

13

solution was lyophilized and redissolved in 5% ACN/ 0.1% FA solution for LC-MS/MS

14

analysis.

15

For enriched glycopeptides from protein, 50 units of PNGase F and the solution were blended

16

homogeneously, and the mixture was disposed at 37 ℃ overnight. In the end, the solution was

17

lyophilized and redissolved in 5% ACN/ 0.1% FA solution for LC-MS/MS analysis.

18

Characterization and MS analysis. The detailed instruments and characterization were

19

displayed in the Supporting Information.

20 21

RESULTS AND DISCUSSION

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Preparation and characteristics of Fe3O4@mSiO2-IDA nanomaterials. The synthetic

23

procedure of Fe3O4@mSiO2-IDA nanomaterials presented in Scheme 1. Firstly, IDA reacted 8 ACS Paragon Plus Environment

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with silane coupling agent (GLYMO) to form GLYMO-IDA compounds by using 65 ℃

2

temperature-controlled water-bath, and then kept the GLYMO-IDA compounds in refrigerator

3

for next step. Secondly, Fe3O4 nanoparticles with 250 nm of mean diameter were synthesized

4

through a hydrothermal method. And then a layer of mesoporous silica was coated on the surface

5

of the Fe3O4 nanoparticles by taking advantage of a kind of surfactant (cetyltrimethylammonium

6

bromide, CTAB) as the structure-directing agent and tetraethyl orthosilicate (TEOS) as the

7

silicon source. Finally, the obtained GLYMO-IDA compounds were grafted onto the inwalls of

8

mesoporous channels. Also, for comparison, we synthesized the hydrophilic magnetic non-

9

mesoporous silica nanomaterials (Fe3O4@nSiO2-IDA) by leaving out the addition of CTAB

10

during the synthetic process of mesopores.

11



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Scheme 1. The synthetic procedure of Fe3O4@mSiO2-IDA nanomaterials.

3

The morphology and crystallography of Fe3O4@mSiO2-IDA nanomaterials were confirmed

4

through diverse characterizations. The single crystal nature of Fe3O4@mSiO2 nanomaterials is

5

shown in Figure S1 (supporting information), the position of the five main characteristic

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diffraction peaks matched well with those previous reports.28,29 In addition, as seen in Figure S2

7

(supporting information), the Fe3O4@mSiO2-IDA nanomaterials could be rapidly dispersed in


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water and separated magnetically, suggesting that the excellent magnetic responsibility of the

2

nanomaterials could simplify the enrichment process.

3 4

Figure 1. TEM images of (A) Fe3O4@mSiO2 nanomaterials; (B, C) Fe3O4@mSiO2-IDA

5

nanomaterials and (D) Fe3O4@nSiO2-IDA nanomaterials.

6

It revealed that the Fe3O4 nanoparticles were well-encapsulated in silica shell from the

7

transmission electron microscopy (TEM) images (Figure 1), which could prevent them from

8

aggregation and be beneficial for them to disperse in buffer solution. Compared with

9

Fe3O4@mSiO2 nanomaterials (Figure 1A), the ordered mesoporous channels of Fe3O4@mSiO2-

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IDA nanomaterials could be observed clearly (Figure 1B-C), which means the mesoporous 11 ACS Paragon Plus Environment


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structure of Fe3O4@mSiO2-IDA was remained after modification of GLYMO-IDA compounds.

2

Figure 1D is the TEM image of the Fe3O4@nSiO2-IDA nanomaterials, however, showing that

3

there is no visible presence of mesopores. The representative IV isotherm with a hysteresis loop

4

of mesoporous nanomaterials defined by IUPAC was obtained (shown in Figure 2) through the

5

N2 adsorption-desorption measurement. The sudden increase of P/P0 from 0.3 to 0.9 indicates

6

that Fe3O4@mSiO2-IDA nanomaterials possessed a uniform pore-size distribution. The

7

calculated Brunauer-Emmett-Teller (BET) surface area and total pore volume of Fe3O4@mSiO2-

8

IDA nanomaterials were about 197.60 m2/g and 0.14 cm3/g, respectively. And from the inset of

9

Figure 2, it could be evaluated that the average pore size of Fe3O4@mSiO2-IDA nanomaterials

10

was about 2.54 nm (BJH mode, Barrett, Joyner, and Halenda) which is suitable for capturing

11

small-size targets. The pore distribution of the Fe3O4@mSiO2 was measured at the same time, as

12

shown in Figure S3 (supporting information), the pore size was evaluated to be 4.15 nm. The

13

pore size after modification of the GLYMO-IDA compounds was smaller than that before, which

14

means GLYMO-IDA compounds was modified on the inwall of mesoporous channels.


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1 2

Figure 2. N2 adsorption-desorption isotherm of Fe3O4@mSiO2-IDA nanomaterials. The inset

3

shows the pore size distribution.

4 5

The chemical composition and structure of the IDA-functionalized nanomaterials was

6

confirmed by Fourier-transform infrared (FTIR) spectroscopy. From Figure 3, the band at 795-

7

798 cm–1 is attributed to the symmetric vibration of Si-O-Si, and the absorption peaks at 1084-

8

1086 cm–1 is ascribed to the asymmetric stretching vibration of Si-O-Si. These above-mentioned

9

bands have showed up in both curves (Figure 3A-B). This suggested that silica was coated on

10

the surface of magnetic nanoparticles. After modification with GLYMO-IDA compounds

11

(Figure 3B), the observed new absorption peak at 2940 cm–1 is assigned to the CH2 stretching

12

vibration and the new absorption band at 1409 cm–1 can be attributed to the existence of CH2-N



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units, which indicates that the successful synthesis and modification of GLYMO-IDA

2

compounds. This result was consistent with the literature.30

3 4

Figure 3. The FTIR spectra of (A) Fe3O4@mSiO2 nanomaterials；(B) Fe3O4@mSiO2-IDA

5

nanomaterials.

6

Moreover, thermal gravimetric analyzer (TGA) was further employed to test the thermal

7

stability and the composition of Fe3O4@mSiO2-IDA nanomaterials. As seen in Figure S4A

8

(supporting information), there is no evident weight loss which indicates the considerable

9

thermal stability of Fe3O4@mSiO2. And from Figure S4 (supporting information), a slight and

10

slower mass loss after 600 ℃ in the curve of Fe3O4@mSiO2 and Fe3O4@mSiO2-IDA can be

11

observed. Taking consideration of the data at 600 ℃, the content of IDA in Fe3O4@mSiO2-IDA 14 ACS Paragon Plus Environment

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nanomaterials was calculated to be about 5.76%. The elemental analysis was displayed in Figure

2

S5 (supporting information), the appearance of nitrogen and carbon in Figure S5E-F indicated

3

that the presence of IDA in Fe3O4@mSiO2-IDA.

4

Optimization of the enrichment performance of Fe3O4@mSiO2-IDA nanomaterials.

5

The enrichment procedure for glycopeptides by Fe3O4@mSiO2-IDA nanomaterials is depicted in

6

Scheme 2, which contains four main steps: enriching, washing, eluting and MS analysis. As seen

7

in Scheme 2, both glycoproteins and non-glycoproteins with large-size can’t come into the

8

mesoporous channels because of the narrow pore size distribution of Fe3O4@mSiO2-IDA

9

nanomaterials. And some non-specific adsorption of non-glycopeptides on the surface of

10

Fe3O4@mSiO2-IDA nanomaterials can be washed away, while glycopeptides can be still retained

11

in the mesopores.

12



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Page 16 of 26

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Scheme 2. The flow chart of the enrichment process for glycopeptides by usingFe3O4@mSiO2-

2

IDA nanomaterials.

3

The enrichment performance of Fe3O4@mSiO2-IDA nanomaterials for glycopeptides was

4

firstly tested by capturing glycopeptides from horeseradish peroxidase (HRP) digestion.

5

According to publications,7,10,31,32 the concentration of acetonitrile (ACN) is quite significance

6

for the glycopeptides enrichment with HILIC materials. Therefore, in this work, ACN /H2O/TFA

7

(80/17/3, v/v/v), ACN/H2O/TFA (89/8/3, v/v/v) and ACN/H2O/TFA (95/2/3, v/v/v) were applied

8

as loading buffer for glycopeptides enrichment. These results were displayed in Figure 4, when

9

using ACN /H2O/TFA (80/17/3, v/v/v) as loading buffer, there are a few glycopeptides with low

10

intensity were observed (Figure 4A). Although ACN/H2O/TFA (95/2/3, v/v/v) as loading buffer

11

achieved the relatively better result (Figure 4C), 89% ACN is enough to obtain the excellent

12

enrichment efficiency, glycopeptide signal-intensities of which are higher (Figure 4B).

13

Therefore, ACN/H2O/TFA (89/8/3, v/v/v) was adopted as loading buffer in the following

14

enrichment experiment. And in another investigation, the number and signal intensity of

15

glycopeptides with Fe3O4@mSiO2-IDA nanomaterials incubation for 20 min were equally well

16

to those acquired for 60 min (Figure S6, supporting information), indicating the rapid adsorption

17

ability of Fe3O4@mSiO2-IDA nanomaterials for glycopeptides.


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1 2

Figure 4. MALDI mass spectra of N-linked glycopeptides enriched from HRP digests with

3

different loading buffer (A) 80% ACN+3%TFA; (B) 89% ACN+3%TFA; (C)95%

4

ACN+3%TFA, using Fe3O4@mSiO2-IDA nanomaterials.

5 6

Application of Fe3O4@mSiO2-IDA nanomaterials in N-linked glycopeptides

7

Enrichment. After optimizing enrichment conditions, there is still one glycopeptide peak could 17 ACS Paragon Plus Environment


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Page 18 of 26

1

be identified (as shown in Figure S7, supporting information) when we further decreased the

2

concentration of HRP digest to 1 fmol/µL, which could be viewed as the lower detection limit of

3

such a strategy. Next, standard protein (bovine serum albumin, BSA) was employed to determine

4

the size-exclusion effect of Fe3O4@mSiO2-IDA nanomaterials. For comparison, the

5

Fe3O4@nSiO2-IDA nanomaterials for removing proteins was carried out. From Figure S8

6

(supporting information), the signal peaks of protein can be obviously detected without

7

enrichment of the Fe3O4@mSiO2-IDA nanomaterials. However, the MALDI mass spectra after

8

enrichment with Fe3O4@mSiO2-IDA nanomaterials (Figure 5) show that the number and peak

9

intensities of glycopeptides maintain pretty stable with the increase of ratio of HRP/BSA from

10

1:0 to 1:200, and no protein signals can be observed from the inset in Figure 5. After enrichment

11

with Fe3O4@nSiO2-IDA nanomaterials, as displayed in Figure S9 (supporting information),

12

there are strong signals of BSA protein (inset, Figure S9, supporting information) can be

13

identified besides 10 glycopeptides. Also, we’ve tested that Fe3O4@mSiO2 nanomaterials and

14

Fe3O4@mSiO2-glymo nanomaterials

15

glycopeptides (results were not displayed). All above results further confirmed that the

16

functional groups were grafted on the Fe3O4@mSiO2-IDA nanomaterials, and the functional

17

groups combined with the mesoporous channels made the novel nanomaterials have a promising

18

efficacy in the aspect of excluding large-size protein for glycopeptide enrichment.

showed

abominable

enrichment

performance

for


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1 2

Figure 5. Enrichment of glycopeptides by Fe3O4@mSiO2-IDA nanomaterials from HRP digests

3

containing different amount of BSA protein with different ratios (w/w). (A) at 1:0; (B) at 1:100;

4

(C) at 1:200 respectively. Glycopeptides are marked with .



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Page 20 of 26

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Furthermore, in order to confirm Fe3O4@mSiO2-IDA nanomaterials have no bias toward all

2

glycopeptides with different glycan types. Immunoglobulin G (Ig G) digestion, which has a

3

different glycan type from HRP, was also regarded as the model sample. As observed in Figure

4

6A, the spectrum was mainly occupied by non-glycopeptides with strong background noise,

5

there was merely one glycopeptide could be detected without enrichment, whereas after

6

enrichment with Fe3O4@mSiO2-IDA nanomaterials, 33 signal peaks of glycopeptide with clear

7

background were observed (shown in Figure 6, Table S1, supporting information).

8 9 10

Figure 6. MALDI mass spectra of glycopeptides from 1 pmol Ig G digestion (A) before and (B) after Fe3O4@mSiO2-IDA nanomaterials enrichment. Glycopeptides are marked with . 20 ACS Paragon Plus Environment

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1

To intensively investigate their feasibility to profile glycosylation, the Fe3O4@mSiO2-IDA

2

nanomaterials were further employed to enrich glycopeptides from complex bio-samples. Human

3

serum as the universal clinical specimen has attracted increasing interests for discovering

4

biomarkers associated with disease diagnosis. In this work, the analysis of glycopeptides from

5

healthy human serum has been performed. The healthy human serum sample was treated with

6

trypsin to form mixture of peptides after reduction and alkylation. And then the enrichment

7

process of human serum was conducted according to the flow chart depicted in Scheme 2.

8

Lastly, the obtained glycopeptides were treated with PNGase F, and the deglycosylated peptides

9

were lyophilized, re-dissolve and sent for nano-LC/MS/MS analysis. Besides the mass increment

10

of 0.9840 Da because of the asparagine transforming to aspartic acid by deamidation, the

11

existence of a consensus N-X-S/T (X can be any amino acid except proline) sequence make the

12

identification of N-linked glycopeptides and glycosylation sites realized. A total of 424

13

glycopeptides in 140 different glycoproteins was finally identified, the detailed information is

14

listed in Table S3 (supporting information). The usage amount of human serum (2 µL) is less

15

than those of previous publications while the number of glycopeptides identified is on the

16

opposite. 10,28

17

CONCLUSIONS

18

In conclusion, the Fe3O4@mSiO2-IDA nanomaterials inherit the virtues of large surface area,

19

excellent hydrophilicity and easy-to-prepare, and could rapidly be separated by external

20

magnetic field, which make this method easily accessible. The Fe3O4@mSiO2-IDA

21

nanomaterials show excellent enrichment performance for both HRP and Ig G digests. And 424

22

glycopeptides assigned to 136 glycoproteins were found from 2 µL human serum. The

23

outstanding ability for glycopeptides enrichment makes it a promising candidate for 21 ACS Paragon Plus Environment


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glycopeptides analysis by MALDI-TOF MS, and indicates its great potential in the aspect of

2

early disease diagnosis.

3

ASSOCIATED CONTENT

4

Supporting Information. This material includes experimental methods, additional data of

5

Fe3O4@mSiO2-IDA characterization and its performance in N-linked glycopeptide enrichment.

6

This material is available free of charge via the Internet at http://pubs.acs.org.

7

AUTHOR INFORMATION

8

Corresponding Authors

9

*Prof. C. H. Deng, E-mail: [email protected], Fax: +86-21-65641740.

10

Notes

11

The authors declare no competing financial interest.

12

ACKNOWLEDGMENT

13

This work was supported by the National Basic Research Priorities Program

14

(2012CB910602, 2013CB911201), the National Natural Science Foundation of China

15

(21425518, 21075022, 21275033, 21105016), Research Fund for the Doctoral Program of

16

Higher Education of China (20110071110007 and 20100071120053).

17 18 19 20 22 ACS Paragon Plus Environment

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