Highly Specific Enrichment of Multi-phosphopeptides by the

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Functional Nanostructured Materials (including low-D carbon)

Highly Specific Enrichment of Multi-Phosphopeptides by the Di-phosphorylated Fructose Modified DualMetal Centered Zirconium-Organic Framework Jiaxi Peng, Huan Niu, Hongyan Zhang, Yating Yao, Xingyun Zhao, Xiaoyu Zhou, Lihong Wan, Kang Xiaohui, and Ren'an Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11138 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Applied Materials & Interfaces

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Highly Specific Enrichment of Multi-Phosphopeptides by the

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Di-phosphorylated Fructose Modified Dual-Metal Centered Zirconium-Organic Framework

3 4 5

a,b

Jiaxi Peng, Huan Niu,a,b Hongyan Zhang,a,b Yating Yao,b Xingyun Zhao,a,b Xiaoyu Zhou,a,b Lihong Wan,a Xiaohui Kang*c and Ren'an Wu*a

6 7 8 9

a

Laboratory of High-Resolution Mass Spectrometry Technologies, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China.

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* E-mail: [email protected]; Fax: +86-411-84379828

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b

University of Chinese Academy of Sciences, Beijing, 100049, China.

12

c

College of Pharmacy, Dalian Medical University, Dalian, 116044, China

13

* E-mail: [email protected]; Fax: +86-411-86110414

14 15

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ACS Paragon Plus Environment

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ABSTRACT: Multisite phosphorylation of a protein, generally occurring in

2

biological processes, plays important roles in the regulation of cellular functions.

3

However, the identification of multi-phosphopeptides especially at low-abundance is

4

a big challenge as the extreme hydrophilicity and poor ionization efficiency of the

5

multi-phosphorylated peptides, restricting the deep inspection of multisite

6

phosphorylation processes. In this study, the highly specific enrichment of

7

multi-phosphorylated peptides was achieved via the modification of the dual-metal

8

centered zirconium-organic framework with the di-phosphorylated fructose. The

9

di-phosphorylated

fructose

modified

dual-metal

centered

zirconium-organic

10

framework (DZMOF-FDP) demonstrated the highly specific affinity to the multiply

11

phosphorylated peptides, with the density functional theory (DFT) calculations

12

explaining the plausible mechanism for multi-phosphopeptides on the DZMOF-FDP.

13

The selective capture of multi-phosphopeptides from mimic samples confirmed the

14

superior performance of the DZMOF-FDP, with the comprehensive comparisons to

15

other modification agents such as orthophosphate and pyrophosphate. A number of

16

1871 multi-phosphorylated peptides captured by DZMOF-FDP from tryptic digests of

17

HeLa cell lysate could be identified, significantly higher than by the pristine DZMOF.

18

The deliberately designed modification with di-phosphorylated fructose for the

19

dual-zirconium centered metal-organic framework materials is suggesting an efficient

20

strategy to develop new enrichment methods in the selective capture of target analytes

21

by judiciously optimizing specific modifiers for adsorbents.

22 23

KEYWORDS: metal-organic framework, multi-phosphopeptide, di-phosphorylated

24

fructose, selective capture, interaction mechanism

25 26

2


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1. INTRODUCTION

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Protein phosphorylation, one of the most essential post-translational modification

3

events, plays a key role in most aspects of cellular functions including signal

4

transduction, gene expression, metabolism, cell growth, and enzymatic regulation.1-2

5

Reversible phosphorylation, usually caused by the opposite action of protein kinases

6

and protein phosphatases, can regulate the “On/Off” states of enzymes as signal

7

switches to modulate cellular processes.3-4 The protein phosphorylation could be

8

modulated by the same kinase on many different amino acid residues, with multisite

9

phosphorylation events occurring progressively (during a single binding event of a

10

kinase to substrate) or distributively (the kinase dissociates from its substrate after

11

each phosphorylation reaction).5 The multisite phosphorylation process of a protein

12

with n sites may generate (2n-1) of phospho-forms and each individual phospho-forms

13

may have distinct biological effects. However, a major difficulty in studying multisite

14

phosphorylation from a system perspective has been the lack of information regarding

15

not only the in-depth mechanisms of these multisite phosphorylation processes but

16

also the dynamic proportions and roles of individual phospho-forms.

17

Most of existing analytical methods have shown the preferences for single

18

phosphorylated peptides but insufficient for multi-phosphopeptides.6-8 The signals of

19

multi-phosphopeptides in mass spectrometry could be severely suppressed by the

20

coexisting mono-phosphopeptides because of their relatively poor ionization

21

efficiency and low content.9-10 Up to date, attempts such as specific metal affinity,11, 10,

22

12

23

were reported for the analysis of multi-phosphopeptides via enrichment procedures.

24

To achieve the comprehensive determination of multisite phosphorylation events for

25

complex biological inspection, the efficiency and the selectivity of the

26

multi-phosphopeptides analysis is yet in urgent demand. Aiming on this purpose, the

27

capture of multi-phosphopeptides would require the enrichment process with: (1)

amine-based functional materials,13 and other specific functional interface,14-16, 7

3



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1

adequate active sites to associate with targets; (2) hydrophilicity to eliminate the

2

severe interference of not only the non-phosphopeptides but also the matrix; (3)

3

specific

4

mono-phosphopeptides.

affinities

to

selectively

distinguish

multi-phosphopeptides

from

5

Metal-organic framework (MOF) as an emerging porous material featuring open

6

metal sites, modifiable interfaces, large specific surface areas, and tunable uniform

7

pore size is demonstrating great potential in sample enrichment.17-20 The inherent

8

active sites of MOFs interacting with phosphate groups have been utilized to absorb,

9

transport, and detect phosphate compounds.21-24 MOFs such as UiO-66, UiO-67,

10

MIL-100 (Fe), and MIL-101 (Fe) have also been applied to capture the

11

phosphopeptides.25-29

12

zirconium-organic framework (DZMOF) possessing inherent Zr-O cluster and

13

immobilized Zr (IV) has been found the high efficiency in the enrichment of both

14

mono- and multi-phosphopetides.30 We noticed that this DZMOF is favor to interact

15

with multi-phosphopeptides as compared with the pristine UiO-66-NH2. To improve

16

the selectivity of MOF materials toward the multi-phosphopeptides, herein, a

17

deliberately designed modification of the DZMOF was carried out to reach the highly

18

specific enrichment of multi-phosphopeptides by specifically denying the binding of

19

mono-phosphorylated peptides (Figure 1a). It was found that the modification of the

20

DZMOF by the hydrophilic fructose-1,6-diphosphate (FDP) exhibited the superior

21

performance in the selective enrichment of the multi-phosphorylated peptides in

22

comparisons with other modifying agents such as orthophosphate and pyrophosphate.

23

The plausible mechanism of the highly selective enrichment of multi-phosphopeptides

24

by the DZMOF with the modification of FDP (DZMOF-FDP) was proposed via the

25

binding energies calculation of DFT for various phosphorus-containing compounds

26

and zirconium sites of DZMOF.

In

our

group,

previously,

4


a

dual-metal

centered

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1 2

Figure 1. (a) Schematic of the preparation of MOF modified with shielding molecules

3

and its application for selective capture of multi-phosphopeptides. TEM images of (b)

4

UiO-66-NH2 and (c) DZMOF. (d) Zeta potential changes of UiO-66-NH2 and

5

DZMOF modified with orthophosphate groups.

6

2. EXPERIMENTAL SECTION

7

2.1. Preparation of DZMOF. DZMOF were prepared according to our previous

8

work. In advance, UiO-66-NH2 was synthesized by a solvothermal method. Briefly,

9

0.5 mmol of ZrCl4, 0.6 mmol of 2-amino-benzenedicarboxylic acid, 15 mmol of

10

benzoic acid and 10 mL of DMF were added into a Teflon-lined stainless-steel

11

autoclave which was then heated in air oven at 120 °C for 24 h. The solid product was

12

immersed with DMF and methanol several times, respectively. Subsequently, 50 mg

13

of UiO-66-NH2 was dispersed into 50 mL of acetonitrile solution with 4 mM POCl3

14

and 4 mM 2,4,6-collidine overnight. After washing with acetonitrile and water for

15

several times, the product was incubated into 10 mL of ZrOCl2 solution (50 mM)

5



1

overnight at ambient temperature. The obtained product, named DZMOF, was washed

2

with water extensively and dried for future use.

3

2.2. Preparation of phosphonate-modified DZMOF. Typically, 10 mg of DZMOF

4

was incubated in 2 mL of fructose-1,6-diphosphate (FDP, 1 mM) solution with

5

vigorous shaking for 30 min at ambient temperature. Then the FDP-modified DZMOF

6

(DZMOF-FDP, the FDP content on DZMOF was about 0.2 µmol/mg and the molar

7

ratio of FDP to Zr was about 3:40) was collected by centrifugation and stepwise

8

washed with 2 mL of NaCl solution (200 mM) once and 2 mL of water twice. The

9

reproducibility of DZMOF-FDP is shown in Figure S5 (TEM images) and Table S3

10

(Zeta potentials). Then DZMOF modified with phosphoric acid (DZMOF-P) or

11

sodium pyrophosphate (DZMOF-PP) was also prepared as the same procedure. Other

12

matrix materials (TiO2 and UiO-66-NH2) were also used to modify with FDP by this

13

procedure for control.

14

2.3. Enrichment of multi-phosphopeptides. In the selective enrichment of

15

multi-phosphopeptides from tryptic digests of α-casein, β-casein, and nonfat milk,

16

respectively, 50 µg of materials were incubated with the peptide mixture in 150 µL of

17

loading buffer (40% acetonitrile (v/v) and 3% TFA (v/v)) for 30 min at room

18

temperature. Then the materials were collected by centrifugation and stepwise rinsed

19

with 150 µL of washing buffer 1 (50% acetonitrile (v/v), 6% TFA (v/v) and 200

20

mmol/L NaCl) and 150 µL of washing buffer 2 (50% acetonitrile (v/v), 0.1% TFA

21

(v/v)). After that, the target peptides were eluted from materials by 10 µL of 10%

22

(w/w) NH3·H2O solution. Then, the eluate was identified by MALDI-TOF MS. To

23

enrich the multi-phosphopeptides from the mixture of α-casein and β-casein protein

24

digests, 120 pmol of α-casein and 40 pmol of β-casein tryptic digests in 150 µL of

25

loading buffer were mixed with 50 µg of MOF materials, and followed the same steps

26

as above.

6


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1

The enrichment of multi-phosphopeptides from tryptic digests of HeLa cell lysate

2

(200 µg) was performed with a material/protein ratio of 20:1 (w/w). Typically, 4 mg

3

of DZMOF-FDP were mixed with 200 µg of HeLa cell lysate digests in 400 µL of

4

loading buffer for 30 min. After washing with 400 µL of washing buffer 1 and 2,

5

respectively, the captured peptides were eluted by 10% (w/w) NH3·H2O solution. The

6

eluted phosphopeptides were then desalted using Ti-IMAC after acidification31 and

7

lyophilized for further Nano-RPLC-ESI-MS/MS analysis.

8

2.4. MALDI-TOF analysis. All MALDI-TOF mass experiments were carried out on

9

AB Sciex 5800 MALDI-TOF/TOF mass spectrometer (AB Sciex, CA) equipped with

10

a pulsed Nd/YAG laser at 355 nm. MALDI-TOF analysis adopted a sample-first

11

method. Briefly, each 0.5 µL of eluate and DHB matrix (25 mg mL−1, 70% ACN and

12

1% H3PO4) was sequentially dropped onto the MALDI stainless steel target for mass

13

analysis.

14

2.5. Nano-RPLC-ESI-MS/MS analysis. The Nano-RPLC-ESI-MS/MS experiments

15

were performed on LTQ-Orbitrap Elite mass spectrometer coupled with Dionex

16

UltiMate 3000 RSLC-nano System (Thermo, San Jose, CA). The lyophilized samples

17

were resuspended into 1% formic acid (FA, v/v) solution and automatically loaded

18

onto a 3 cm C18 trap column (200 µm i.d.) at a flow rate of 3 µL/min. The separation

19

was performed on a 15 cm C18 column (150 µm i.d.) with a flow rate of 600 nL/min.

20

Water with 0.1% FA (buffer A) and 80% acetonitrile with 0.1% FA (buffer B) were

21

used as mobile phase to generate a 176 min gradient, set as follows: 0% B for 10 min,

22

0‒3% B for 3 min, 3‒30% B for 135 min, 30‒45% B for 15 min, 45‒99% B for 2 min

23

and 99% B for 11 min. The LTQ-Orbitrap Elite mass spectrometer was operated in a

24

positive, data dependent MS/MS acquisition mode. The ion transfer capillary

25

temperature was set at 275 °C and the spray voltage was 2.5 kV. The full mass scan

26

acquired in the Orbitrap mass analyzer was from m/z 350 to 1650 with a resolution of

27

60000 and the top 20 parent ions with charge states ≥ 2 from the full scan were 7



1

fragmented by collision-induced dissociation (CID) with 35% normalized collision

2

energy. The dynamic exclusion function was set as follows: repeat count 1, repeat

3

duration 30 s, and exclusion duration 90 s.

4

2.6. Database searching. The acquired MS/MS spectra were searched by Mascot

5

(version 2.4.1) against a human protein database from Uniprot. The search parameters

6

were set as follows: trypsin as the specific proteolytic enzyme with a maximum of

7

two missed cleavages allowed; the carbamidomethylation of cysteine (C) was set as a

8

fixed modification; the oxidation of methionine (M), phosphorylation of serine (S),

9

threonine (T), and tyrosine (Y) were specified as variable modifications;

10

precursor-ion mass error tolerance was set to 20 ppm, and fragment-ion mass error

11

tolerance was set to 0.8 Da. Peptides with the false discovery rate (FDR) < 0.01 and

12

with a minimum score of 20 were accepted as confident identifications.

13

2.7. Computational details. Molecular geometries of the studied species were

14

optimized with the Gaussian 09 program32 at the DFT level using the B3LYP

15

functional.33-34 The 6-31G(d) basis set was used for non-metal atoms, and Zr atom

16

was treated by the LANL2DZ effective core potential (ECP) and the associated basis

17

sets. Normal-coordinate analyses were performed to verify the geometrically

18

optimized stationary points. In the calculations of binding energy, the energies of the

19

phosphorus-containing compound moiety and the remained MOF part in their optimal

20

geometry of MOF-phosphorus-containing compounds complex were evaluated via

21

single-point calculations. Such single-point energies of the fragments and the energy

22

of MOF-phosphorus-containing compounds complex were used to estimate the

23

binging energy. To obtain more reliable energies, the 6-31+G(d,p) basis set for

24

non-metal atoms and the same basis set as that geometric optimization for Zr atom

25

were used, and the basis set superposition error (BSSE) correction was included.35

26

3. RESULTS AND DISCUSSION 8


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1

3.1. Enrichment of phosphopeptides by MOF modified with orthophosphate

2

groups. DZMOF, which possesses dual metal centers of both inherent Zr-O cluster

3

and immobilized Zr (IV), has been reported to have high selectivity in the enrichment

4

of phosphopeptides in our previous work.30 To improve the selectivity of

5

multi-phosphopeptides, the enrichment materials were further modified with more

6

hydrophilic species to enhance the multi-phosphopeptides retention and decrease the

7

nonspecific binding by hydrophobic interactions.10 Phosphoric acid as a hydrophilic

8

compound has been introduced to occupy the surface sites of metal oxide materials

9

and improve their selectivity toward multi-phosphopeptides significantly.36 As an

10

attempt, UiO-66-NH2 and DZMOF (Figure 1b and c) were chosen as matrix materials

11

to conveniently modify with orthophosphate groups by an immersion process.

12

Compared to the original matrix materials, the zeta potential of orthophosphate groups

13

modified UiO-66-NH2 (UiO-66-NH2-P) and DZMOF (DZMOF-P) showed different

14

degrees of decline (Figure 1d), illustrating the effectiveness of immersion for

15

modifying phosphate species. The more reduced zeta potential of DZMOF-P also

16

reflected that there were more metal sites on the surface of DZMOF for binding with

17

orthophosphate groups, which might be beneficial for the enrichment of

18

phosphopeptides. Both UiO-66-NH2-P and DZMOF-P were then performed to enrich

19

phosphopeptides from tryptic digests of α-casein, respectively.

20

As shown in MALDI spectra, the signals of mono-phosphopeptides declined

21

obviously and the proportion of multi-phosphopeptides increased after capture by

22

orthophosphate modified MOFs (Figure 2). The introduction of orthophosphate

23

groups on MOF surface was effective to prevent a part of mono-phosphopeptides to

24

bind with metal sites and improve the selectivity for multi-phosphopeptides in a

25

certain extent. Moreover, three additional multi-phosphopeptides (α16, α20, and α25,

26

illustrated in Table S1) were captured by DZMOF-P and the intensities of

27

multi-phosphopeptides especially including more than two phosphate groups were 9



1

higher than extracted by UiO-66-NH2-P. It might be ascribed to the fact that the

2

matrix material of DZMOF-P has more active metal centers to likely bind with

3

multi-phosphopeptides. This result shows the promising potential of DZMOF as an

4

available matrix for surface regulation in multi-phosphopeptide enrichment.

5 6

Figure 2. MALDI-TOF mass spectra of α-casein tryptic digests (0.2 nmol) after

7

enrichment by (a) DZMOF, (b) UiO-66-NH2-P, and (c) DZMOF-P. “*” denoted

8

dephosphorylated fragments.

9

3.2. Comparison of different modifiers for DZMOF. To further enhance the

10

selectivity of DZMOF toward multi-phosphopeptides, two different approaches were

11

tried: increasing the quantity of modified phosphoric acid and altering the variety of

12

phosphate compounds on the surface of DZMOF. To enhance the quantity of

13

orthophosphate groups of DZMOF-P, the concentration of initial phosphoric acid

14

solution was increased from 1 to 10 mM. The obtained products were applied to

15

capture the tryptic digests of α-casein. Unfortunately, after modified with more

16

orthophosphate groups on DZMOF, the pattern of mass spectrum was similar with the

17

enrichment result extracted by pristine DZMOF-P and the intensity of peaks was even 10


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1

lower (Figure S1). It illustrates that the increased orthophosphate groups were not

2

efficient for selective substitution of multi-phosphopeptides and simultaneously

3

decreased the interaction with all types of phosphopeptides. On the other hand,

4

another two modifiers (sodium pyrophosphate and FDP) were chosen to regulate the

5

surface property of DZMOF (Figure S2). As shown in Figure 3, the proportions of

6

multi-phosphopeptides captured by DZMOF modified with sodium pyrophosphate

7

(DZMOF-PP) and FDP (DZMOF-FDP) were improved. Meanwhile, the proportions

8

of mono-phosphopeptides were gradually declining. It was worth noting that the

9

intensities of phosphopeptides with over two phosphate groups (α19, α20, α21, α22,

10

and α25) were obviously enhanced, which could significantly contribute to identify

11

multi-phosphopeptides in mass spectrometric analysis.16

12

Furthermore, the signal to noise ratios (S/N) of multi-phosphopeptides captured by

13

DZMOF-PP and DZMOF-FDP were almost enhanced than by DZMOF and

14

DZMOF-P, while the S/N of mono-phosphopeptides captured by DZMOF-PP and

15

DZMOF-FDP were lower (Figure S3). Obviously, the number and intensity of

16

multi-phosphopeptides extracted by DZMOF-FDP were increased more efficiently

17

than by DZMOF-P and DZMOF-PP. The tightly coupled FDP on the surface of

18

DZMOF which might be unable to be substituted by mono-phosphopeptides in the

19

incubation fluid made the interaction of mono-phosphopeptides with DZMOF-FDP

20

rather weak. When the phosphate groups on phosphopeptides increased, which was

21

beneficial for competitively replacing the FDP molecules on the surface of DZMOF,

22

the signals of multi-phosphopeptides extracted by DZMOF-FDP were enhanced. Thus

23

the hydrophilic FDP molecule modified on the surface of DZMOF showed great

24

potential in the application of multi-phosphopeptide enrichment.

11



1 2

Figure 3. MALDI-TOF mass spectra of α-casein tryptic digests (80 pmol) after

3

enrichment by (a) DZMOF-P, (b) DZMOF-PP, and (c) DZMOF-FDP. “*” denoted

4


5

To explore the interaction mechanism between phosphonate-modified DZMOF

6

and phosphopeptides, the binding energies between various phosphorus-containing

7

compounds and zirconium sites of DZMOF, which reflect the interaction strength,

8

were computed by DFT calculations (Table 1). The binding energies of phosphoric

9

acid with inherent zirconium (Zr1, Figure 4a) and with modified zirconium (Zr2)

10

were ‒184.67 and ‒237.28 kcal/mol, respectively, while the binding energies of a

11

typical mono-phosphopeptide (Asn-pSer-Ala) with two single zirconium sites

12

(Zr1/Zr2) were ‒180.73 and ‒245.28 kcal/mol, respectively. Therefore, the modified

13

zirconium shows stronger absorption ability to phosphopeptides which is consistent

14

with previous experimental results.30 In order to explore the effectiveness of modifiers

15

on DZMOF for improving the multi-phosphopeptide selectivity, we also investigate

16

the binding ability of pyrophosphoric acid, FDP, and multi-phosphopeptide with

17

DZMOF. The computational results was that the binding energies between 12


Page 12 of 24

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1

pyrophosphoric acid and two single zirconium sites (Zr1/Zr2) were ‒399.40 and ‒

2

441.22 kcal/mol, respectively, while the binding energy of FDP or a typical

3

multi-phosphopeptide (pSer-Glu-pSer) with two zirconium sites (Zr1-Zr2) was ‒

4

486.40 (Figure 4b) or ‒488.89 kcal/mol (Figure 4c), respectively.

5

Table 1. The binding energies of phosphorus-containing compounds with zirconium

6

sites of DZMOF. Binding energy (kcal/mol) Coordination Phosphoric

Pyrophosphoric

acid

acid

sites

FDP

Mono-

Multi-

phosphopeptide

phosphopeptide

(Asn-pSer-Ala)

(pSer-Glu-pSer)

Zr1

‒184.67

‒399.40

/

‒180.73

/

Zr2

‒237.28

‒441.22

/

‒245.28

/

Zr1-Zr2

/

/

‒486.40

/

‒488.89

7

8 9

Figure 4. Schematic of (a) inherent zirconium (Zr1) and modified zirconium (Zr2) in

10

DZMOF, (b) interaction between FDP and two zirconium sites in DZMOF, and (c)

11

interaction between multi-phosphopeptide and two zirconium sites in DZMOF.

12

Zirconium, oxygen, carbon, nitrogen, and phosphorus atoms are denoted by yellow,

13

red, grey, blue, and pink, respectively. 13



Page 14 of 24

1

By contrast, the interaction of DZMOF with multi-phosphopeptide was

2

obviously stronger than with phosphoric acid and with mono-phosphopeptide, thereby

3

showing the improving selectivity toward multi-phosphopeptides by phosphoric acid

4

modified DZMOF. This result is consistent with the experimental results (Figure 2c

5

and 3a). However, the similar binding energies of phosphoric acid and

6

mono-phosphopeptide with each zirconium sites illustrated that the pre-adsorbed

7

phosphate groups of DZMOF-P could also be replaced by mono-phosphopeptides

8

under a certain probability. On the other hand, due to the exceptionally high binding

9

energy between single zirconium site and pyrophosphoric acid, the zirconium sites

10

modified

by

pyrophosphoric

acid

were

difficult

to

bind

with adequate

11

multi-phosphopeptides, which decreased the recovery of multi-phosphopeptides after

12

enrichment by DZMOF-PP as shown in Figure 3b. Importantly, the binding energy of

13

the two zirconium sites with FDP was much larger than that of the two single

14

zirconium sites with mono-phosphopeptide, but slightly smaller than that of those

15

with multi-phosphopeptide. This confirmed that the interaction between FDP and

16

DZMOF might effectively shield the mono-phosphopeptides and selectively contact

17

with the multi-phosphopeptides, which showed a good agreement with the enrichment

18

experiment treated by DZMOF-FDP (Figure 3c).

19

To further study the superiority of DZMOF-FDP in multi-phosphopeptide

20

enrichment, a series of DZMOF materials were applied to incubate with a mixture of

21

α-casein (120 pmol) and β-casein (40 pmol) protein digests. When the affinity probe

22

DZMOF modified with phosphate species, the signals of multi-phosphopeptides were

23

significantly enhanced and the relative intensities of the mono-phosphopeptides had

24

different

25

multi-phosphopeptides were increased obviously after enrichment with DZMOF-P

26

while the proportions of mono-phosphopeptides were slightly decreased. In addition,

27

partial of mono-phosphopeptide signals were eliminated after treated with

degrees

of

decline

(Figure

5).

Briefly,

14


the

proportions

of

Page 15 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

DZMOF-PP and the proportions of multi-phosphopeptides were enhanced. In

2

particular,

3

mono-phosphopeptides and captured large numbers of multi-phosphopeptides.

4

Therefore,

5

multi-phosphopeptides compared with DZMOF, DZMOF-P, and DZMOF-PP.

DZMOF-FDP

DZMOF-FDP

nearly

exhibited

eliminated

the

highest

the

interferences

selective

ability

of

toward

6 7

Figure 5. The relative abundance of phosphorylated peptides from the mixture of

8

α-casein and β-casein protein digests extracted by DZMOF, DZMOF-P, DZMOF-PP,

9

and DZMOF-FDP, respectively.

10

The sensitivity of DZMOF-FDP was further investigated by gradual dilution of

11

β-casein digests. A loading solution (150 µL) with 40 pmol, 4 pmol, 0.4 pmol, and

12

0.04 pmol tryptic digests of β-casein was incubated with DZMOF-FDP (50 µg),

13

respectively. Through performing the routine enrichment protocol, the mass spectra

14

clearly showed (Figure S4) the multi-phosphopeptide (β3) was enriched without any

15

interference. Even when the absolute quantity of β-casein digests was as low as 0.04

16

pmol in the loading solution, the signal of β3 (S/N=274) was still obviously observed 15



Page 16 of 24

1

in the mass spectrum. These results indicated that the identification of

2

multi-phosphopeptides was highly sensitive by using DZMOF-FDP.

3

3.3.

4

multi-phosphopeptides. To examine the importance of matrix materials for the

5

modification of the robust FDP, TiO2 and UiO-66-NH2 were chosen as represented

6

matrix materials. It is effective for TiO2 modified with FDP (TiO2-FDP) to improve

7

the selectivity of multi-phosphopeptide, but the intensities of high level

8

phosphorylated peptides especially with three or more phosphorylation sites were

9

extremely low (Figure 6a). Although the FDP molecules on the surface of TiO2 inhibit

10

the non- and mono-phosphopeptides, the inherent metal sites of TiO2 may not be

11

suitable for enrichment of high level phosphorylated peptides. It has been reported

12

that multi-phosphopeptides adsorbed on metal oxides (such as TiO2 and ZrO2), which

13

possessing strong adsorption sites, are hard to be desorbed.36 On the other hand, the

14

mass

15

UiO-66-NH2-FDP were quite low and the mono-phosphopeptides were not

16

completely eliminated (Figure 6b). It seems that UiO-66-NH2-FDP with monotonous

17

metal site and randomly distributed FDP molecules on the surface does not have the

18

ability to differentiate mono- and multi-phosphopeptides. It is interesting that the

19

selective enrichment toward global multi-phosphopeptides was performed only on the

20

specific

21

multi-phosphopeptides not only relies on the properties of surface modifiers but also

22

on the inherent characters of matrix materials.

Importance

spectrum

interface

of

matrix

showed

of

the

materials

intensities

DZMOF-FDP.

In

for

of

selective

enrichment

phosphopeptides

other

words,

16


the

extracted

enrichment

of

by

of

Page 17 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 6. MALDI-TOF mass spectra of α-casein tryptic digests (80 pmol) after

3

enrichment

4


5

3.4. Highly specific enrichment of multi-phosphopeptides from complex samples.

6

Since few phosphoprotein in body fluid has been developed for disease diagnosis in

7

clinical practice,37 more effective isolation and identification for dynamic phorylated

8

peptides/proteins are urgently demanded. The performance of multi-phosphopeptide

9

enrichment by DZMOF-FDP for real complex sample was investigated by applying

10

tryptic digests of nonfat milk and HeLa cell lysate. The multi-phosphopeptides

11

extracted by DZMOF-FDP from nonfat milk digests were clearly shown in the mass

12

spectrum while there were almost mono-phosphopeptides captured by DZMOF

13

dominating the spectrum (Figure 7). There were 17 multi-phosphopeptides captured

14

by DZMOF-FDP from nonfat milk digests, which was benefit from the efficient

15

selectivity of DZMOF-FDP. Moreover, the DZMOF-FDP was applied to capture the

16

multi-phosphopeptides from the tryptic digests of HeLa cell lysate. In total, 2669

by

(a)

TiO2-FDP

and

(b)

UiO-66-NH2-FDP.

17


“*”

denoted


Page 18 of 24

1

phosphopeptides including 1871 multi-phosphopeptides (70.1%) were feasibly

2

identified after pretreatment with DZMOF-FDP, while 3257 phosphopeptides

3

including 605 multi-phosphopeptides (18.6%) were identified after pretreatment with

4

DZMOF (Figure 8). The selective ability of DZMOF-FDP for multi-phosphopeptides

5

was improved by more than three times compared with the result obtained by

6

DZMOF.

7

phosphopeptides

8

multi-phosphopeptides captured by DZMOF-FDP represented 41.4, 13.9, and 14.7%

9

of the total identified phosphopeptides. In comparison, only corresponding 14.2, 1.8,

10

and 2.6% were captured by DZMOF (Table S2). This significant improvement shows

11

the great potential of DZMOF-FDP in the discovery of novel multiple

12

phosphorylation sites.

Interestingly, with

di-phosphopeptides,

four

or

more

tri-phosphopeptides,

phosphorylation

sites

and

among

1871

13 14

Figure 7. MALDI-TOF mass spectra of phosphorylated peptides in nonfat milk

15

digests

16


extracted

by

(a)

DZMOF

and

(b)

DZMOF-FDP.

18


“*”

denoted

Page 19 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 8. Distribution proportions of mono- and multi-phosphopeptides extracted by

3

DZMOF (left) and DZMOF-FDP (right) from tryptic digests of HeLa cell lysate.

4

3. CONCLUSIONS

5

A novel functionalization method for MOF has been developed for highly

6

specific enrichment of multi-phosphopeptide. The hydrophilic fructose molecular

7

with two phosphate groups (FDP) was demonstrated as an efficient modifier to

8

regulate the surface properties of MOF. Moreover, by controlling the variables of

9

modifiers and matrixes, respectively, it was found that not only the surface modifiers

10

but also the matrix materials made great contributions to the specific enrichment of

11

multi-phosphopeptides. The dual-metal centered zirconium-organic framework

12

modified

13

multi-phosphopeptides

14

DZMOF-FDP toward multi-phosphopeptides could mainly be attributed to two main

15

reasons. The first is that the application of hydrophilic and flexible FDP molecules to

16

DZMOF generates a functional complex with metal-rich matrixes and diphosphate

17

pre-coordinated interface. The strong interaction between FDP and metal sites of

18

DZMOF produces a high anti-interference performance for eliminating the

19

non-phosphorylated peptides and mono-phosphopeptides. The second is the robust

20

binding between multi-phosphopeptides and DZMOF through the enrichment

21

processes, which could substitute the FDP on the surface of DZMOF. Furthermore,

with

FDP

(DZMOF-FDP) from

mimic

showed samples.

the

best

The

superior

19


selectivity

toward

specificity

of


1

DZMOF-FDP was successfully applied to selectively enrich multi-phosphopeptides

2

from the complex biological mixtures (nonfat milk and HeLa cell lysate digests). This

3

may promote the application of MOF materials in multi-phosphopeptide enrichment

4

for a comprehensive understanding of biological multiple phosphorylation process.

5 6

ASSOCIATED CONTENT

7

Supporting Information

8

The Supporting Information is available free of charge on the ACS Publications

9

website at DOI:

10

Additional data of reagents and materials, samples preparation, figures of

11

MALDI-TOF mass spectra, Zeta potential, signal to noise ratios, and tables of

12

identified phosphopeptides and distribution.

13

AUTHOR INFORMATION

14

Corresponding Authors

15

*E-mail: [email protected]

16

*E-mail: [email protected]

17

Author Contributions

18

All authors have given approval to the final version of the manuscript.

19

Notes

20

The authors declare no competing financial interest.

21

ACKNOWLEDGMENTS

20


Page 20 of 24

Page 21 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

The financial supports from the National Natural Science Foundation of China (Nos.

2

21675156, 21375125 and 21704011), the Instrument Developing Project of the

3

Chinese Academy of Sciences (YZ201503), the CAS Key Laboratory Foundation of

4

Separation Science for Analytical Chemistry, and the Innovation Program (DICP

5

TMSR201601) of Science and Research from the Dalian Institute of Chemical

6

Physics are greatly acknowledged.

7

REFERENCES

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

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Highly Specific Enrichment of Multi-phosphopeptides by the

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