Facile Preparation of Titanium(IV)-Immobilized Hierarchically Porous

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Facile Preparation of Titanium (IV)-immobilized Hierarchically Porous Hybrid Monoliths Haiyang Zhang, Junjie Ou, Yating Yao, Hongwei Wang, Zhongshan Liu, Yinmao Wei, and Mingliang Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00242 • Publication Date (Web): 18 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

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Analytical Chemistry

1 2

Facile Preparation of Titanium (IV)-immobilized Hierarchically

3

Porous Hybrid Monoliths

4 5

Haiyang Zhanga, b, c, Junjie Oua,*, Yating Yaoa, c, Hongwei Wanga, c, Zhongshan Liua, c,

6

Yinmao Weib,*, Mingliang Yea,*

7 8

a

9

Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of

10

b

11

of Education, College of Chemistry and Materials Science, Northwest University,

12

Xi'an 710069, China

13

c

Key Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry

University of Chinese Academy of Sciences, Beijing 100049, China

14

1

ACS Paragon Plus Environment


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ABSTRACT

16

Hierarchically porous materials have become a key feature of biological materials

17

and have been widely applied for adsorption or catalysis. Herein, we presented a new

18

approach to directly prepare a phosphate-functionalized hierarchically porous hybrid

19

monolith (HPHM), which simultaneously contained mesopores and macropores. The

20

design

21

vinylsilsesquioxanes (vinylPOSS) and vinylphosphonic acid (VPA) by adding

22

degradable polycaprolactone (PCL) additive. The phosphate groups could be directly

23

introduced into the hybrid monoliths. This approach was simple and time-saving, and

24

overcame the defect of rigorous complex process of preparing traditional

25

Ti4+-immobilized metal ion affinity chromatography (IMAC) materials. The specific

26

surface area of optimal hybrid monolith could reach 502 m2/g obtained by nitrogen

27

adsorption/desorption measurements, which originated from the degradation of PCL.

28

Meanwhile, the characterization of scanning electron microscopy (SEM) and mercury

29

intrusion porosimetry (MIP) also suggested that the macropores existed in the hybrid

30

monoliths. The size of macropores could be controlled by the content of PCL in the

31

polymerization mixture. The prepared Ti4+-IMAC HPHMs exhibited high adsorption

32

capacity (63.6 mg/g for pyridocal 5’-phosphatemonohydrate), excellent enrichment

33

specificity (tryptic digest of β-casein/BSA at a molar ratio of 1:1000) and sensitivity

34

(tryptic digest of 5 fmol of β-casein). Moreover, the Ti4+-IMAC HPHMs provided

35

effective enrichment ability of low-abundance phosphopeptides from human serum

36

and Hela cell digests.

was

based

on

the

copolymerization

of

2


polyhedral

oligomeric

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37

INTRODUCTION

38

As one of crucial tools for signaling in cellular networks, protein phosphorylation

39

has become a major regulatory machinery in regulating many complicated biological

40

processes.1,2 Nowadays, mass spectrometry (MS)-based techniques have become a

41

preferred technique for the characterization of protein phosphorylation. Owing to the

42

co-existence of abundant non-phosphopeptides, the direct MS analysis was not able to

43

identify low abundant phosphopeptides in the complex peptide mixtures generated

44

from protein digest. Therefore, it was indispensable to specifically isolate subsets of

45

phosphopeptides from biological samples prior to MS analysis.

46

Biological matter still sets important guiding principles for materials science, and

47

various materials have been developed for selective enrichment of phosphopeptides.3-8

48

Among them, nanoparticles, polymer-based monoliths and microspheres are the most

49

commonly used materials. Nanoparticles, such as titanium dioxide,9,10 mesopores

50

silica,11 magnetic nanoparticles12,13 and metal-organic framework nanoparticles,14,15

51

showed high enrichment specificity and sensitivity for phosphopeptides in biological

52

samples. However, these materials usually required a few steps to modify their

53

surfaces with specific function, which was troublesome and time-consuming.

54

Titanium (IV) immobilized polymeric monoliths16-22 were also remarkable materials

55

for enrichment of phosphopeptides. Although monolithic supports had the advantages

56

such as high porosity and convective mass transport, relatively low specific surface

57

area of monoliths,23 which was less than tens of square meters per gram (m2/g) and

58

limited their application. In 2013, Heck and Zou et al24 developed a titanium (IV)

59

immobilized mono-dispersed microsphere and applied for immobilized metal ion

60

affinity chromatography (Ti4+-IMAC). This kind of Ti4+-IMAC microsphere also

61

exhibited excellent enrichment performance of phosphopeptides. However, the

62

fabrication process of Ti4+-IMAC microsphere also required several steps such as

63

formation of mono-dispersed polystyrene microspheres, coupling of phosphonate

64

groups onto the mono-dispersed microspheres and so on, which was really

65

complicated and time-consuming. Therefore, the design and synthesis of a novel 3



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66

IMAC material for high selectivity of enriching phosphopeptides is still attracting

67

attention.

68

Hierarchical porous materials were key materials used for adsorption or catalysis,

69

which were predominantly understood as a structural feature and have been in the

70

focus of scientific research for more than ten years.25-28 These materials

71

simultaneously contain a percolating pore structure and a continuous matrix structure

72

that may combines micro- (< 2 nm), meso- (2-50 nm) and macropores (> 50 nm).

73

Such multilevel porous architectures confer unique properties to materials depending

74

on the combination of pore sizes, in which micro- and mesopores generate a large

75

specific surface area, providing main functional sites, while macropores can improve

76

the mass transfer in the application process, increasing the accessibility of the active

77

sites.25,29-32 As a result, hierarchically porous materials exhibit lower backpressures,

78

higher permeability and better performance in separation and life science.33-36

79

However, there are few reports on hierarchically porous materials used for

80

phosphopeptides analysis.37,38

81

In this work, we developed a novel Ti4+-IMAC hybrid monolithic material with

82

hierarchical structure, which simultaneously consisting of micropores, mesopores and

83

macropores.

84

vinylphosphonic acid (VPA) as monomers, polycaprolactone (PCL) as additive and

85

tetrahydrofuran (THF) as single solvent were employed to prepare Ti4+-IMAC

86

hierarchically porous hybrid monoliths (HPHMs) via thermal-initiated free radical

87

polymerization. The preparation process was very simple and accessible, and the

88

resulting Ti4+-IMAC HPHMs with hierarchical structure demonstrated high binding

89

capacity of small molecules, high selectivity and detection sensitivity for

90

phosphopeptides in different biological samples.

Polyhedral

oligomeric

vinylsilsesquioxanes

(vinylPOSS)

and

91 92

EXPERMENTAL SECTION

93

Materials.

94

VinylPOSS was obtained from Hybrid Plastics, Inc (Hattiesburg, MS, USA). VPA 4


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(97%), PCL (average Mn~10,000), pyridoxal 5’-phosphate hydrate (≥ 98%), sodium

96

chloride (NaCl), trifluoroacetic acid (TFA), bovine serum albumin (BSA), β-casein,

97

protease inhibitor Cocktail (for use with mammalian cell and tissue extracts),

98

phosphatase inhibitor (1 mM NaF and 1 mM Na3VO4), TPCK treated trypsin, urea,

99

iodoacetamide (IAA), 1,4-dithiothreitol (DTT) and 2,5-dihydroxyl benzoic acid (DHB)

100

were obtained from Sigma (St Louis, MO, USA). 2,2-Azobisisobutyronitrile (AIBN)

101

and titanium sulfate were gotten from Shanghai chemical Plant (Shanghai, China).

102

Tetrahydrofuran (THF), hydrochloric acid (HCl) and methanol were of analytical

103

grade, and obtained from Tianjin Kermel Chemical Plant (Tianjin, China).

104

RPMI-1640 cell culturing medium was purchased from Gibco Invitrogen Corporation

105

(Carlsbad, CA). Formic acid (FA) was obtained from Fluka (Buches, Germany).

106

Acetonitrile (ACN, HPLC grade) and ammonia-water (NH3•H2O) were purchased

107

from Merck (Darmstadt, Germany). The water used in all experiments was doubly

108

distilled and purified by Milli-Q system (Millipore Inc., Milford, MA, USA).

109

Preparation of Phosphate-functionalized HPHMs.

110

VinylPOSS, VPA and PCL were dissolved in THF by sonication for 5 min, and the

111

detail composition of prepolymerization mixtures and porous properties of

112

phosphate-functionalized HPHMs were listed in Table 1. After AIBN was added into

113

the solution, the solution was transferred into an ampoule, and then placed in the

114

liquid nitrogen to solidify the solution. Meanwhile, the ampoule was vacuumized by a

115

vacuum pump, and the bottleneck of ampoule was sealed by using a butane torch at

116

the same time. After that the ampoule was put in 60 oC water bath for 24 h. The

117

obtained materials were washed with THF for three times to remove residual

118

monomer, and then dried in air atmosphere and put in the vacuum at 60 oC for 12 h.

119

Subsequently, the materials were degraded by placing them in vials containing 1 M

120

HCl in water/methanol (6/4, v/v) solution. The vials were sealed with parafilms and

121

heated for 3 days at 70 oC water bath to ensure complete hydrolysis of PCL. The

122

obtained bulk monoliths (7.0 mm diameter × 8.0 mm length, cylinder) were rinsed

123

with water and methanol repeatedly. Finally, the hybrid monoliths were dried in a

124

vacuum oven at 60 oC overnight. 5



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125

Preparation of Ti4+-IMAC HPHMs.

126

Ti4+ was immobilized on phosphate-functionalized HPHMs by using the phosphate

127

groups existed in the hybrid monoliths as chelating agents. Prior to immobilization of

128

Ti4+, the monoliths were ground to amorphous granules (more than tens of

129

micrometers) in order to weigh easily. Titanium sulfate aqueous solution (100 mg/mL)

130

was prepared in centrifuge tube, and then the hybrid monoliths were added. The

131

centrifuge tube was put on the rolling incubator for 12 h to guarantee entire binding.

132

Then the hybrid monoliths were washed by water and 200 mM NaCl aqueous solution

133

(containing 0.1% TFA) to remove dissociative Ti4+.

134

Phosphopeptides Enrichment Using Ti4+-IMAC HPHMs

135

Before enrichment of phosphopeptides, 5 mg of Ti4+-IMAC HPHMs were placed in

136

a centrifuge tube and equilibrated by loading solution (80% ACN, 6% TFA). At the

137

same time, the tryptic peptide digest was mixed in loading solution (80% ACN, 6%

138

TFA) with identical volumes. Then the tryptic peptide digest in loading solution was

139

transferred into the centrifuge tube in which Ti4+-IMAC HPHMs existed. The mixture

140

was gently incubated at room temperature for 30 min. After removing the supernatant,

141

the Ti4+-IMAC HPHMs were washed twice with 200 µL of washing solution (50%

142

ACN, 6% TFA, 200 mM NaCl) and another washing solution (30% ACN, 0.1% TFA)

143

orderly to remove the non-specifically bound peptides. The captured phosphopeptides

144

were eluted twice by 100 µL of 10% NH3•H2O. After centrifugation, the supernatants

145

containing phosphopeptides were collected in the centrifuge tube. The obtained

146

phosphopeptides were lyophilized using a Speed-Vac (Thermo SPD SpeedVac) and

147

then stored at -30 oC for further LC-MS/MS analysis.

148

Instruments and Methods.

149

Fourier-transformed infrared spectroscopy (FT-IR) characterization was carried out

150

on Thermo Nicolet 380 spectrometer (Nicolet, Wisconsin, USA) using KBr pellets,

151

containing approximate 1 mg sample and 100 mg KBr. The microscopic morphology

152

and energy dispersive spectrometer (EDS) of hybrid monolithic materials were

153

obtained by scanning electron microscopy (SEM, JEOL JSM-5600, Tokyo, Japan).

154

The specific surface areas were calculated from nitrogen adsorption/desorption 6


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155

measurements of dry bulk monoliths using a Quadrasorb SI surface area analyzer

156

(Quantachrome, Boynton Beach, USA). Samples were vacuumized and treated with

157

120 oC for 6 h before nitrogen adsorption/desorption analysis. The specific surface

158

areas were calculated via the Brunauer-Emmett-Teller (BET) method, and the pore

159

size

160

Barrett-Joyner-Halenda (BJH) method. The total pore volumes were determined at

161

P/P0 = 0.99. UV-vis spectra were recorded on a V550 spectrophotometer (JASO,

162

Japan). PoreMasre GT-60 (Quantachrome Boynton Beach, USA) was used to measure

163

macropores size distribution of hybrid monoliths by mercury intrusion porosimetry

164

(MIP). Thermogravimetric (TG) data were collected on Pyris 1 TGA (Perkin Elmer,

165

USA).

distributions

were

determined

from

adsorption

isotherm

by

the

166

The phosphopeptides were enriched without lyophilization according to the

167

previous described method. The mixture of phosphopeptides in 10% NH3•H2O (0.5

168

µL) and the matrix (0.5 µL of 25 mg/mL DHB in ACN/H2O/H3PO4 = 70/29/1, v/v/v)

169

were spotted on the MALDI plate, and analyzed by an AB Sciex 5800

170

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

171

Nd/YAG laser at 355 nm in linear positive ion mode.

172 173

RESULTS AND DISCUSSION

174

Preparation and Characterization of Ti4+-IMAC HPHMs

175

Polyhedral oligomeric silsesquioxanes (POSS), as a kind of organic-inorganic

176

hybrid nanocomposite, has been widely incorporated into various materials,39-41 which

177

can result in significant improvement in physical and mechanical properties due to the

178

reinforcement at the molecular level and the inorganic framework’s ceramic-like

179

properties.42,43 Poly(lactide) (PLA), a chemically degradable polymer, has been

180

employed to synthesize nanoporous polymers,44-46 and the structure of PCL is similar

181

with PLA. Thus, PCL can also be degraded in basic or acidic conditions. As shown in

182

Scheme 1, the preparation process of Ti4+-IMAC HPHMs was illustrated. The hybrid

183

monoliths were firstly formed via thermal-initiated free radical polymerization of 7



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Page 8 of 28

184

vinylPOSS and VPA as monomers, PCL as nonreactive additive and THF as the

185

solvent. In this way, the phosphate groups were directly introduced into the hybrid

186

monoliths. The approach overcame the defect of the immobilization of phosphate

187

groups into other Ti4+-IMAC materials. The PCL was trapped in the bulk monoliths in

188

the polymerization process and could be degraded after a treatment of acidic solution.

189

Although it took about five days to synthesize Ti4+-IMAC HPHMs, the operation

190

process was simple and labor-saving.

191

Figure 1a and c presented the nitrogen sorption isotherm and pore size distribution

192

of hybrid monolith II, respectively, which was prepared with equal molar of VPA and

193

vinylPOSS (vinyl group molar ratio of 1:8) and adding 25% (w/w) PCL. The results

194

clearly indicated the existence of both micropores and mesopores, and the hybrid

195

monolith exhibited pore volume of 0.32 cm3/g and high surface area of 502 m2/g. The

196

size of mesopores was smaller than 10 nm based on BJH analysis of the adsorption

197

isotherm (Figure 1c).The hybrid monolith VI was fabricated by adding more VPA into

198

the prepolymerization solution (vinyl group molar ratio of 1:4), which also contained

199

25% (w/w) PCL. As shown in Figure 1b and d, the specific surface area of hybrid

200

monolith VI was slightly decreased to 473 m2/g, and the size of mesopores was the

201

same as that of hybrid monolith II (less than 10 nm). Further increasing VPA into the

202

prepolymerization solution (vinyl group molar ratio of VPA/vinylPOSS, 1:2) to

203

prepare the hybrid monolith X, it could also be seen that the specific surface area was

204

further decreased to 360 m2/g as shown in Table 1. These results suggested that the

205

specific surface areas of hybrid monoliths decreased by adding more VPA into the

206

prepolymerization solution. This phenomenon was possibly related to either

207

homopolymerization of VPA or copolymerization of VPA and vinylPOSS. First, the

208

poly(vinylphosphonic acid) (poly(VPA)) was formed via homopolymerization of VPA,

209

which

210

phosphate-functionalized hybrid monolithic materials. The poly(VPA) was a kind of

211

linear polymer, which had relatively low specific surface areas.47,48 Thus, the

212

ingredient with relatively low specific surface areas would cause to decrease the

213

specific surface areas of hybrid monoliths. Meanwhile, part of phosphonate ligands

was

then

coupled

with

vinylPOSS

or

8


monoliths

to

form

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214

was embedded in the skeleton of the HPHM, which was not accessible. Second, a

215

VPA molecule was linked with two molecules of vinylPOSS monomer via

216

copolymerization reaction, which would form a flexible chain. Intertwining might

217

occur between these flexible chains, resulting in the decrease of specific surface areas

218

of hybrid monoliths. Therefore, the specific surface areas of hybrid monoliths

219

decreased with an increase of the content of VPA monomer in the prepolymerization

220

mixture. As shown in Figure S1, the Si and P were uniformly distributed on the

221

surface of hybrid monoliths. It was deduced that the phosphate groups were also

222

evenly distributed in the hybrid monoliths.

223

In order to verify the effect of PCL additive on the porous properties of hybrid

224

monoliths, different amounts of PCL (from 16.7 to 31.8%) were added into the

225

prepolymerization solution, in which the vinyl group molar ratio of VPA/vinylPOSS

226

maintained 1:4 (shown in Table 1, monoliths III-VIII). It can be seen that the specific

227

surface areas of these monoliths after degradation of PCL were ranged in 406-473

228

m2/g, and the pore volumes were in the range of 0.26-0.35 cm3/g, which were

229

measured by nitrogen adsorption/desorption measurements. The size of mesopores in

230

monolith VI was also less than 10 nm (Figure 1d). For comparison, monolith III was

231

prepared without adding PCL, and the specific surface area reached 460 m2/g, in

232

which the micropores specific surface area was 225 m2/g by t-plot method, while the

233

mesopores specific surface area was 215 m2/g. Although the introduction of PCL did

234

not have remarkable effect on the specific surface areas and pore volumes, the

235

specific surface areas of hybrid monoliths without degradation of PCL were far lower

236

than those after degradation of PCL as shown in Table 1. It was clearly shown that the

237

specific surface area of hybrid monolith VI prior to degradation of PCL was only 174

238

m2/g, in which the micropores specific surface area reached 83 m2/g by the t-plot

239

method, while the mesopores specific surface area was only 91 m2/g. However, the

240

specific surface area reached 473 m2/g after degradation of PCL. The micropores

241

specific surface area reached 273 m2/g by the t-plot method, while the mesopores

242

specific surface area was 200 m2/g. Further increasing the content of PCL to 31.8%,

243

the result was the same, in which the specific surface area of monolith VIII increased 9



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244

from 154 to 406 m2/g after degradation of PCL. Additionally, this phenomenon could

245

be observed on the monoliths II and X, which were prepared with other vinyl group

246

molar ratios of VPA/vinylPOSS (1:8 and 1:2, respectively, Table 1). The specific

247

surface area of monolith II increased from 195 to 502 m2/g after degradation of PCL,

248

while the specific surface area of monolith X increased from 97 to 360 m2/g after

249

degradation of PCL. These results indicated that PCL additive could occupy the

250

monolithic scaffold when the copolymerization of vinylPOSS and VPA occurred,

251

which was not be washed out by solvents, but only removed by acidic hydrolysis to

252

generate nanopores.

253

The characterization results of scanning electron microscopy (SEM) and mercury

254

intrusion porosimetry (MIP) further demonstrated that the hybrid monoliths prepared

255

with PCL possessed macroporous structure. As shown in Figure 2 and S2, the SEM

256

micrograph of monolith VI showed that the macropores clearly appeared when the

257

content of PCL reached 25%. However, as shown in Figure S3, the macropores could

258

not be observed in the monolith VI prior to degradation of PCL, also indicating that

259

the PCL could not be washed out by solvent. When the content of PCL was further

260

increased to 31.8%, the size of macropores in hybrid monolith VIII became larger

261

(Figure 2c and f) than that in hybrid monolith VI (Figure 2b and e). However, there

262

were not distinct macropores observed from the SEM micrograph of monolith III,

263

which was prepared without adding PCL (Figure 2a, d and S2a). It was indicated that

264

PCL also had effect on formation of macropores. Meanwhile, the macropores could

265

also be observed from Figure S4, as the content of PCL reached 25%, and the content

266

of VPA was changed (monolith II). The results of MIP characterization further

267

testified that macropores clearly appeared in monolith VI (Figure 3a), and the size of

268

macropores in monolith VIII became larger (nearly 1 µm) further increasing the

269

content of PCL to 31.8% (Figure 3b). The results of nitrogen adsorption/desorption

270

measurements, SEM and MIP proved that PCL could facilitate to form not only

271

nanopores, but also macropores in the framework of hybrid monoliths. Meanwhile,

272

the size of macropores can be controlled by the content of PCL in the

273

prepolymerization mixture. In a word, the phosphate-functionalized hybrid monoliths 10


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274

(II, VI and VIII) simultaneously possessed micropores, mesopores and macropores,

275

indicating hierarchically porous structure. Therefore, the phosphate-functionalized

276

HPHMs were facilely prepared via free radical polymerization following the

277

degradation of PCL.

278

The Fourier-transformed infrared spectroscopy (FT-IR) characterization was

279

performed to inspect the formation of hybrid monolith by using vinylPOSS and VPA

280

as the monomers, as shown in Figure 4. It could be clearly observed that the peak

281

signals at 3030, 3070 cm-1 (assigned to C-H stretching vibrations from CH=CH2) and

282

1600 cm-1 (assigned to C=C stretching vibration) existed in the spectrum of

283

vinylPOSS monomer (Figure 4a). After the formation of hybrid monoliths, the peaks

284

at 3030 and 3070 cm-1 almost disappeared (Figure 4d). Compared to the FT-IR

285

spectrum of VPA (Figure 4b), the strong signal peak at 930 cm-1, which was assigned

286

to the symmetrical stretching vibration of phosphate radical, could be clearly observed

287

in the FT-IR spectrum of hybrid monolith (Figure 4d). It was indicated that phosphate

288

groups existed in hybrid monolith. However, other characteristic adsorption peaks of

289

phosphate (such as 1161 and 1003 cm-1, which assigned to the stretching vibration

290

P=O and P-O, respectively) were covered by the characteristic adsorption peaks of

291

vinylPOSS. A weak signal at 1730 cm-1 implied the appearance of carbonyl (C=O) in

292

PCL (Figure 4c), which still existed in hybrid monolith. This result suggested that

293

PCL could not be absolutely degraded by acidic condition, and part of PCL might be

294

wrapped in the skeleton. The existence of PCL would not have influence on the

295

properties of hybrid monoliths. Meanwhile, thermal stability of hybrid monolith was

296

investigated by TG, as shown in Figure S5. A significant mass loss began at 300 oC

297

and continued to 600 oC due to the pyrolysis of organic moieties under nitrogen

298

atmosphere. This result indicated that the obtained HPHMs exhibited satisfactory

299

thermal stability. Meanwhile, the residue of hybrid monolith VI before immobilization

300

of Ti4+ was 44.1%, while it was 62.8% after immobilization of Ti4+. It was due to the

301

residue contained titanium compounds, which could not sublimate at 800 oC. This

302

result also indicated that Ti4+ was successfully immobilized on hybrid monolith.

303 11



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Properties of Ti4+-IMAC HPHMs in Absorption and Enrichment

305

Pyridocal 5’-phosphatemonohydrate as a coenzyme plays an important role in all

306

transamination reactions and some decarboxylation or deamination reactions of amino

307

acids. Due to the existence of phosphate group, pyridocal 5’-phosphatemonohydrate

308

can be absorbed by Ti4+-IMAC materials based on the chelation between Ti4+ and

309

phosphate group. Therefore, it was selected to evaluate the adsorption capacity of

310

phosphate-functionalized HPHMs. Prior to use, the prepared phosphate-functionalized

311

HPHMs were firstly chelated with titanium ions to obtain Ti4+-IMAC HPHMs. The

312

curve of the adsorption isotherm of pyridocal 5’-phosphatemonohydrate was shown in

313

Figure 5A. Three kinds of Ti4+-IMAC materials, including Ti4+-IMAC hybrid

314

monolith without any macropores (monolith III), Ti4+-IMAC HPHM (monolith VI)

315

and Ti4+-IMAC microspheres (12 µm, diameter) prepared according to the reference24,

316

were employed to absorb pyridocal 5’-phosphatemonohydrate. It could be seen that

317

the adsorption equilibration (○) was achieved on monolith VI within 30 min, while it

318

would take 60 min to reach the adsorption equilibration (■) on monolith III. This

319

result demonstrated that macropores existed in monolith VI could facilitate to increase

320

the mass transfer rate. Although the adsorption equilibration (▲) using Ti4+-IMAC

321

microspheres mentioned above could be achieved within several minutes, the

322

adsorption

323

microspheres was only 49.7 mg/g, as shown in Figure 5b. Compared to the adsorption

324

capacity of Ti4+-IMAC microspheres, the adsorption capacity of monolith VI was

325

higher, which could reach 63.6 mg/g. However, the adsorption capacity of monolith

326

III was only 38.8 mg/g, which was far lower than those of other two materials. These

327

results illustrated that although the micropores and mesopores surface areas of

328

monolith VI (273 and 200 m2/g, respectively) were similar with those of monolith III

329

(225 and 215 m2/g, respectively), the macropores existed in the hybrid monoliths

330

could enhance the mass transfer rate and shorten the equilibration time. Meanwhile,

331

its high specific surface area could provide much more active sites and improve the

332

adsorption capacity. It was deduced that this kind of Ti4+-IMAC HPHMs was

333

applicable to enrich small molecules containing phosphate groups.

capacity

for

pyridocal

5’-phosphatemonohydrate

12


on

Ti4+-IMAC

Page 13 of 28

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334

Considering the hierarchically porous property, monolith VI was selected for

335

evaluation of the ability of Ti4+-IMAC HPHMs in the enrichment of phosphopeptides.

336

Due to the low level of phosphopeptides in a complex biological sample, the

337

sensitivity and selectivity of Ti4+-IMAC HPHMs were two key parameters for the

338

enrichment performance. A standard phosphoprotein (β-casein) tryptic digest was

339

used to evaluate its performance. The tryptic digest of 1 µg of β-casein (100 fmol)

340

was incubated with 5 mg of Ti4+-IMAC HPHM in loading solution, and then the

341

bound peptides were eluted by 10% NH3•H2O after washing away nonspecific

342

peptides with washing solutions. Finally, 0.5 µL of the eluted solution was deposited

343

on the MALDI target for MALDI-TOF MS analysis. As shown in Figure S6a, the

344

tryptic digest of β-casein was directly analyzed by MALDI-TOF MS without any

345

enrichment. It could be seen that the signals of the phosphopeptides were severely

346

suppressed by many non-phosphopeptides, and the peaks of phosphopeptides could

347

not be clearly observed. However, the peaks of three expected phosphopeptides could

348

be clearly seen with a clear background after enrichment with HPHM as shown in

349

Figure S6b, along with one of its dephosphorylated counterparts was also detected.

350

Meanwhile, the non-phosphopeptides peaks nearly completely disappeared, which

351

indicated that this kind of Ti4+-IMAC HPHMs had high affinity toward

352

phosphopeptides. The sensitivity of phosphopeptides enriched by the Ti4+-IMAC

353

HPHMs with MALDI-TOF MS detection was also performed by using different

354

contents of β-casein tryptic digest as shown in Figure S6c and d. The results showed

355

that the phosphopeptides in β-casein tryptic digest whose content reached 10 fmol

356

could be still detected by MALDI-TOF MS after enriching with the monolith VI.

357

Even though the total amount of β-casein tryptic digest reached as low as 5 fmol, one

358

peak of phosphopeptide could still be identified in the spectrum. Therefore, it was

359

deduced that the Ti4+-IMAC HPHMs could be used to process trace amount of

360

samples. Furthermore, a mixture of β-casein and BSA tryptic digest as test sample

361

was used to evaluate the selectivity of the Ti4+-IMAC HPHMs for enrichment of

362

phosphopeptides. When the molar ratio of β-casein to BSA tryptic digest was 1:500,

363

three peaks of characteristic phosphopeptides in β-casein tryptic digest could be easily 13



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

364

detected by MALDI-TOFMS after enrichment using monolith VI, as shown in Figure

365

S7a. Even when the molar ratio of β-casein to BSA tryptic digest decreased to 1:1000,

366

three expected phosphopeptides could still be clearly identified in spite of the

367

interference signal from non-phosphopeptides was slightly increased as shown in

368

Figure S7b. These results illustrated that the prepared Ti4+-IMAC HPHMs had high

369

selectivity for the capture of phosphopeptides from a complex peptide mixture.

370 371

Application of Ti4+-IMAC HPHMs in Enrichment of Phosphopeptides from

372

Human Serum and Tryptic Digest of Human Hela Cells

373

To further demonstrate the feasibility of Ti4+-IMAC HPHMs in selective

374

enrichment of low abundance of phosphopeptides from practical biosamples, human

375

serum was selected as a real sample. As shown in Figure 6a, only one MS signal

376

intensity of phosphopeptides appeared owing to the low abundance of

377

phosphopeptides and high salt content, while several non-phosphopeptides with

378

intense MS signal appeared in the spectrum. Nonetheless, after treatment with

379

Ti4+-IMAC HPHM, four peaks of phosphopeptides with higher MS intensities could

380

be distinctly detected as shown in Figure 6b. The detail information of four

381

phosphopeptides from human serum was shown in Table S1. This result revealed that

382

the prepared Ti4+-IMAC HPHMs were capable of highly selective trapping of

383

phosphopeptides from a complicated biological sample.

384

Encouraged by its excellent performance in enrichment of phosphopeptides, the

385

phosphopeptides from human Hela cell digests were also enriched by Ti4+-IMAC

386

HPHMs. The 100 µg of human Hela cell digests and 5 mg of monolith VI were used

387

each time, and three technical repeats were performed in parallel. The obtained

388

phosphorylated peptides were then analyzed by LC-MS/MS. For comparison,

389

Ti4+-IMAC microspheres were also used to capture phosphopeptides from human

390

Hela cell digests under its optimal condition at the same time.24 The 1851, 1923 and

391

1894 of unique phosphopeptides were identified from 100 µg of Hela cell digests after

392

enriching by monolith VI, respectively, while only 1733, 1686 and 1683 of unique

393

phosphopeptides were identified after enriching by Ti4+-IMAC microspheres, 14


Page 14 of 28

Page 15 of 28

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394

respectively.

Meanwhile,

the

enrichment

specificities

(the

percentages

of

395

phosphopeptides identified) after enriched by Ti4+-IMAC HPHM or Ti4+-IMAC

396

microspheres were all higher than 97.0%. These results indicated that the prepared

397

Ti4+-IMAC HPHMs exhibited excellent enrichment specificity and selectivity towards

398

to phosphopeptides and could be applied to comprehensive phosphoproteome

399

analysis.

400 401

CONCLUSIONS

402

In summary, a novel Ti4+-IMAC HPHM was successfully synthesized via

403

thermal-initiated free radical polymerization of vinylPOSS and VPA by introducing

404

degradable PCL additive. Phosphate groups could be directly introduced into the

405

hybrid monoliths, and the obtained hybrid monoliths possessed hierarchical structures

406

after degradation of PCL, which simultaneously possessed macropores, mesopores

407

and micropores. The nanopores came from the degradation of PCL additive in the

408

hybrid monolithic framework as well as the nanopores originated from the

409

copolymerization of vinylPOSS and VPA due to the steric hindrance could provide

410

large specific surface areas and lots of active phosphate sites. Meanwhile, the content

411

of PCL could also affect the formation of macropores, which could enhance the mass

412

transfer rate. Compared to the methods to prepare other Ti4+-IMAC microspheres and

413

nanomaterials, the method to prepare this kind of HPHMs was easy and time-saving

414

due to without extra steps to introduce the phosphate groups. The resulting Ti4+-IMAC

415

HPHMs exhibited excellent adsorption capacity, enrichment specificity and sensitivity

416

for phosphopeptides. Furthermore, this kind of Ti4+-IMAC HPHMs was used to

417

selectively enrich phosphopeptides from human serum and Hela cell digest, and the

418

results showed great practicability in identifying low-abundance phosphopeptides

419

from complicated biological samples.

420 421

AUTHOR INFORMATION

422

Corresponding Author 15



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

423

*E-mail:

[email protected]

(J.J.

Ou);

[email protected]

424

[email protected] (Y.M. Wei)

425

Notes

426

The authors declare no competing financial interest.

Page 16 of 28

(M.L.

Ye);

427 428

SUPPORTING INFORMATION AVAILABLE

429

Additional information as noted in text. This material is available free of charge via

430

the internet at http://pubs.acs.org.

431 432

ACKONWLEDGMENTS

433

Financial support is gratefully acknowledged from the China State Key Basic

434

Research Program Grant (2016YFA0501402) and the National Science Fund for

435

Distinguished Young Scholars (21525524) to M. Ye, as well as the National Natural

436

Sciences Foundation of China (No. 21575141) to J. Ou.

437 438

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

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Greis, K. D. ACS Appl. Mater. Inter. 2015, 7, 11155-11164. (11) Chen, Y.; Li, D.; Bie, Z.; He, X.; Liu, Z. Anal. Chem. 2016, 88, 1447-1454. (12) Jabeen, F.; Najam-Ul-Haq, M.; Rainer, M.; Guzel, Y.; Huck, C. W.; Bonn, G. K. Anal. Chem. 2015, 87, 4726-4732. (13) Xiong, Z.; Zhang, L.; Fang, C.; Zhang, Q.; Ji, Y.; Zhang, Z.; Zhang, W.; Zou, H. J. Mater. Chem. B 2014, 2, 4473. (14) Chen, Y.; Xiong, Z.; Peng, L.; Gan, Y.; Zhao, Y.; Shen, J.; Qian, J.; Zhang, L.; Zhang, W. ACS Appl. Mater. Inter. 2015, 7, 16338-16347. (15) Chen, L.; Ou, J.; Wang, H.; Liu, Z.; Ye, M.; Zou, H. ACS Appl. Mater. Inter. 2016, 8, 20292-20300. (16) Hou, C.; Ma, J.; Tao, D.; Shan, Y.; Liang, Z.; Zhang, L.; Zhang, Y. J Proteome Res 2010, 9, 4093-4101. (17) Feng, S.; Pan, C.; Jiang, X.; Xu, S.; Zhou, H.; Ye, M.; Zou, H. Proteomics 2007, 7, 351-360. (18) Krenkova, J.; Foret, F. Anal. Bioanal. Chem. 2013, 405, 2175-2183. (19) Wang, S. T.; Wang, M. Y.; Su, X.; Yuan, B. F.; Feng, Y. Q. Anal. Chem. 2012, 84, 7763-7770. (20) Cernigoj, U.; Gaspersic, J.; Fichtenbaum, A.; Lendero Krajnc, N.; Vidic, J.; Mitulovic, G.; Strancar, A. Anal. Chim. Acta. 2016, 942, 146-154. (21) Saeed, A.; Maya, F.; Xiao, D. J.; Najam-ul-Haq, M.; Svec, F.; Britt, D. K. Adv. Funct. Mater. 2014, 24, 5790-5797. (22) Krenkova, J.; Lacher, N. A.; Svec, F. Anal. Chem. 2010, 82, 8335-8341. (23) Liu, Z.; Ou, J.; Lin, H.; Liu, Z.; Wang, H.; Dong, J.; Zou, H. Chem. Commun. 2014, 50, 9288-9290. (24) Zhou, H.; Ye, M.; Dong, J.; Corradini, E.; Cristobal, A.; Heck, A. J.; Zou, H.; Mohammed, S. Nat. Protoc. 2013, 8, 461-480. (25) Verboekend, D.; Nuttens, N.; Locus, R.; Van Aelst, J.; Verolme, P.; Groen, J. C.; Perez-Ramirez, J.; Sels, B. F. Chem. Soc. Rev. 2016, 45, 3331-3352. (26) Schwieger, W.; Machoke, A. G.; Weissenberger, T.; Inayat, A.; Selvam, T.; Klumpp, M.; Inayat, A. Chem. Soc. Rev. 2016, 45, 3353-3376. (27) Zhang, Y.; Zhang, Y.; Burke, J. M.; Gleitsman, K.; Friedrich, S. M.; Liu, K. J.; Wang, T. H. Adv. Mater. 2016, 28, 10630-10636. (28) Lopez-Orozco, S.; Inayat, A.; Schwab, A.; Selvam, T.; Schwieger, W. Adv. Mater. 2011, 23, 2602-2615. (29) Sai, H.; Tan, K. W.; Hur, K.; Asenath-Smith, E.; Hovden, R.; Jiang, Y.; Riccio, M.; Muller, D. A.; Elser, V.; Estroff, L. A.; Gruner, S. M.; Wiesner, U. Science 2013, 341, 530-534. (30) Schneider, D.; Mehlhorn, D.; Zeigermann, P.; Karger, J.; Valiullin, R. Chem. Soc. Rev. 2016, 45, 3439-3467. (31) Huang, X.; Yu, H.; Chen, J.; Lu, Z.; Yazami, R.; Hng, H. H. Adv. Mater. 2014, 26, 1296-1303. (32) Hasell, T.; Zhang, H.; Cooper, A. I. Adv. Mater. 2012, 24, 5732-5737. (33) Konishi, J.; Fujita, K.; Nakanishi, K.; Hirao, K.; Morisato, K.; Miyazaki, S.; Ohira, M. J. Chromatogr. A 2009, 1216, 7375-7383. (34) López-Noriega, A.; Arcos, D.; Izquierdo-Barba, I.; Sakamoto, Y.; Terasaki, O.; Vallet-Regí, M. Chem. Mater. 2006, 18, 3137-3144. (35) Sanchez, C. m.; Belleville, P.; Popalld, M.; Nicolea, L. Chem. Soc. Rev. 2011, 40, 696-753. (36) Hartmann, M.; Schwieger, W. Chem. Soc. Rev. 2016, 45, 3311-3312. 17



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(37) Nischang, I.; Causon, T. J. TrAC Trends Anal. Chem. 2016, 75, 108-117. (38) Sun, M. H.; Huang, S. Z.; Chen, L. H.; Li, Y.; Yang, X. Y.; Yuan, Z. Y.; Su, B. L. Chem. Soc. Rev. 2016, 45, 3479-3563. (39) Nischang, I.; Bruggemann, O.; Teasdale, I. Angew. Chem. Int. Ed. 2011, 50, 4592-4596. (40) Ou, J.; Liu, Z.; Wang, H.; Lin, H.; Dong, J.; Zou, H. Electrophoresis 2015, 36, 62-75. (41) He, H. B.; Li, B.; Dong, J. P.; Lei, Y. Y.; Wang, T. L.; Yu, Q. W.; Feng, Y. Q.; Sun, Y. B. ACS Appl. Mater. Inter. 2013, 5, 8058-8066. (42) Cordes, D. B.; Lickiss, P. D.; Rataboul, F. Chem. Rev. 2010, 110, 2081-2173. (43) Zhang, H.; Ou, J.; Liu, Z.; Wang, H.; Wei, Y.; Zou, H. Anal. Chem. 2015, 87, 8789-8797. (44) Seo, M.; Hillmyer, M. A. Science 2012, 336, 1422-1425. (45) Saba, S. A.; Mousavi, M. P.; Buhlmann, P.; Hillmyer, M. A. J. Am. Chem. Soc. 2015, 137, 8896-8899. (46) Seo, M.; Kim, S.; Oh, J.; Kim, S. J.; Hillmyer, M. A. J. Am. Chem. Soc. 2015, 137, 600-603. (47) Higashihara, T.; Fukuzaki, N.; Tamura, Y.; Rho, Y.; Nakabayashi, K.; Nakazawa, S.; Murata, S.; Ree, M.; Ueda, M. J. Mater. Chem. A 2013, 1, 1457-1464. (48) Markiewicz, K. H.; Seiler, L.; Misztalewska, I.; Winkler, K.; Harrisson, S.; Wilczewska, A. Z.; Destarac, M.; Marty, J. D. Polym. Chem. 2016, 7, 6391-6399.

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523

Figure captions

524

Scheme 1. Schematic approach for preparation of Ti4+-IMAC hierarchically porous

525

hybrid monoliths (HPHMs).

526

Figure 1. (a, b) Nitrogen sorption isotherms and (c, d) mesopores size distributions of

527

hybrid monoliths based on BJH analysis of the adsorption. Hybrid monoliths (a,c) II

528

and (b, d) VI.

529

Figure 2. SEM micrographs of hybrid monoliths (a, d) III, (b, e) VI and (c, f) VIII.

530

Figure 3. Macropores size distributions of hybrid monoliths (a) VI and (b) VIII by

531

MIP measurement.

532

Figure 4. FT-IR spectra of (a) vinylPOSS, (b) VPA, (c) PCL and (d) HPHM.

533

Figure 5. (a) Adsorption kinetics and (b) adsorption isotherms for pyridoxal

534

5’-phosphate hydrate by Ti4+-IMAC microspheres (▲), monolith III (■) and monolith

535

VI (○).

536

Figure 6. MALDI-TOF mass spectra of human serum (a) before and (b) after

537

enrichment by Ti4+-IMAC HPHM. (*) indicates phosphopeptides.

538

19



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

539 540 541

542 543

Scheme 1.

544 545 546 547 548 549 550

20


Page 20 of 28

Page 21 of 28

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


551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

Figure 1.

21



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

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583

Figure 2.

22


Page 22 of 28

Page 23 of 28

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


584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607

Figure 3.

23



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

608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625

Figure 4.

626 627 628 629 630 631 632 633 634 635 24


Page 24 of 28

Page 25 of 28

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


636

637 638

Figure 5.

639 640 641 642 643 644 645 646 647 648 649 650 651 652 653

25



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

654 655 656

Figure 6.

657

26


Page 26 of 28

Page 27 of 28

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


658 659

Table 1. Detail composition of prepolymerization mixtures and porous properties of

660

phosphate-functionalized hybrid monoliths Monolith

VPA a

PCL (%) b

before degradation I II III IV V VI VII VIII IX X

1:8 1:8 1:4 1:4 1:4 1:4 1:4 1:4 1:2 1:2

Pores volume (cm3/g)

Surface area (m2/g)

0 25.0 0 16.7 21.0 25.0 28.6 31.8 0 25.0

after degradation 501 502 440 409 438 473 413 406 368 360

195 ˗c ˗ 174 ˗ 154 97

0.32 0.32 0.28 0.26 0.28 0.35 0.27 0.29 0.25 0.29

661

a

The quantity of VPA was based on the vinyl group molar ratio of VPA/vinylPOSS.

662

b

All PCL content (w/w) was the ratio of the weight of PCL to the total weight of

663

vinylPOSS and PCL.

664

c

665

adsorption/desorption measurements.

The surface areas of hybrid monoliths had not been measured by nitrogen

666 667 668 669 670 671 672 673 674 675 676 677 678 27



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

679

for TOC only

680 681

28


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Facile Preparation of Titanium(IV)-Immobilized Hierarchically Porous

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