TiO2 nanowires from wet-corrosion synthesis for peptide sequencing

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Biological and Medical Applications of Materials and Interfaces

TiO2 nanowires from wet-corrosion synthesis for peptide sequencing using laser desorption/ionization time-of-flight mass spectrometry Mira Kim, Jong-Min Park, Tae Gyeong Yun, Joo-Yoon Noh, Min-Jung Kang, and Jae-Chul Pyun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03804 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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ACS Applied Materials & Interfaces

TiO2 nanowires from wet-corrosion synthesis for peptide sequencing using laser desorption/ionization time-of-flight mass spectrometry

Mira Kima,#, Jong-Min Parka,#, Tae Gyeong Yuna, Joo-Yoon Noha, Min-Jung Kangb,*, and Jae-Chul Pyuna,*

aDepartment

of Materials Science and Engineering, Yonsei University, 134

Shinchon-dong, Seodaemun-gu, Seoul 03722, Korea; Tel) +82 2 2123 5851 / Fax) +82 2 312 5375 Email) [email protected] bKorea

Institute of Science and Technology (KIST), Seoul 02792, Korea

#

These two authors contributed equally to this work. *

To whom correspondence should be addressed.

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

ABSTRACT

In this work, TiO2 nanowires synthesized from a wet-corrosion process were presented for peptide sequencing by photocatalytic reaction with UV radiation. For the photocatalytic decomposition of peptides, the peptide sample was dropped on a target plate containing synthesized TiO2 nanowire zones and UV-irradiated. Subsequently, the target plate was analyzed by laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry using the synthesized TiO2 nanowires as a solid matrix. The feasibility of peptide sequencing based on the photocatalytic reaction with the synthesized TiO2 nanowires was demonstrated using six types of peptides GHP9 (G1-H-P-Q-G2-K1-K2-K3-K4, 1006.59 Da), BPA-1(K1-S1-L-E-N-S2-Y-G1-G2-G3-K2-K3-K4, 1394.74 Da), PreS1(F1-G-A-N1-SN2-N3-P1-D1-W-D2-F2-N4-P2-N5, 1707.68 Da), HPQ peptide-1 (G-Y-H-P-Q-R-K, 884.45 Da), HPQ peptide-2 (K-R-H-P-Q-Y-G, 884.45 Da), and HPQ peptide-3 (R-Y-H-P-Q-G-K, 884.45 Da). The identification of three different peptides with the same molecular weights was also demonstrated by using the synthesized TiO2 nanowires for their photocatalytic decomposition, as well as for LDI-TOF mass spectrometry as a solid-matrix.

Keywords: TiO2 nanowire, wet-corrosion, peptide sequencing, anatase, photocatalytic decomposition

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1. Introduction Matrix-assisted

laser

desorption/ionization

time-of-flight

(MALDI-TOF)

mass

spectrometry has been widely used for the analysis of high-molecular-weight biomolecules such as proteins and nucleic acids.1 MALDI-TOF mass spectrometry is advantageous in mild ionization without inducing the fragmentation of analyte molecules with

organic

matrix

molecules.

However,

the

application

of

MALDI-TOF

mass

spectrometry to small molecules is difficult because the mass peaks of the fragmented matrix molecules are irreproducible in the m/z range less than 1 kDa. Additionally, inhomogeneous dried crystals of the sample and matrix molecules on the target plate cause the issue of “sweet spots.” Usually, the sweet spot referred to a part of a dried sample spot where the sample ions could be quantitatively prepared according to the sample concentration.2-3 For the quantitative analysis of small molecules, various types of nanostructures have been

developed

as

solid

matrices

to

replace

the

organic

matrices

in

laser

desorption/ionization time-of-flight (LDI-TOF) mass spectrometry.4-6 The nanostructures were usually composed of carbon materials, such as carbon nanotubes,7 graphenes,8-9 and diamond-like carbons;10 semiconductor materials,5,

11

such as ZnO, SiC, SnO2, and

TiO2; and metal nanoparticles, such as Ag,12-13 Pt,14 and Au,15-16 among others.17-18 In addition, organic matrices with thin parylene films have been also used to analyze small molecules without fragmented matrix molecules in the m/z range less than 1 kDa, as well as for the quantitative analysis of amino acids and antibiotics.19-23 Among the above inorganic nanostructures, TiO2 structures, including thin films,24-25 nanoparticles,26-27 and nanowires28-29 were frequently used for the LDI mass spectrometric analysis of small

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molecules, because the photocatalytic activity of TiO2 nanostructures could effectively ionize the sample upon irradiation using an ultraviolet (UV) laser for LDI-TOF mass spectrometry.30-34 Recently, the fabrication of TiO2 nanowires for LDI-TOF mass spectrometry by the wet corrosion of a TiO2 plate was reported, which could be used effectively for the quantitative measurement of small molecules such as amino acids.28-29 In this work, a TiO2 nanowire plate were prepared by the wet-corrosion process and used for peptide sequencing. Peptides are small proteins, usually comprising fewer than 40 amino acids, and peptide sequencing refers to the determination of the amino acid sequence from the N- to C-terminals. Such sequencing was often performed by inducing chemical reactions that sequentially detach amino acids from the N- or C-terminal of the peptide and then analyzing the detached amino acids using liquid chromatography.35-39 Such chemical methods required as many repetitions of the detachment reaction depending on the number of amino acid residues in a given peptide. MALDI-TOF mass spectrometry was also used for peptide sequencing. In MALDI-TOF mass spectrometry for peptide sequencing, the peptide was first ionized and detached from the target plate, and then fragmented by collisions with accelerated gas molecules (often He, N2, or Ar) during the flight to the detector. Such a process is called collision-induced dissociation (CID).40-42 The CID method can also distinguish different molecules of identical molecular weights. MALDI-TOF mass spectrometry can determine the peptide sequence through a single CID process, regardless of the number of component amino acids. However, this method requires an expensive tandem mass spectroscopy instrument for the CID process to induce peptide fragmentation.43-44

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In this work, TiO2 nanowires synthesized through the wet-corrosion process was used for peptide sequencing by the two-step reaction: (1) fragmentation of a peptide by photocatalytic decomposition with UV radiation and (2) analysis of the peptide fragments by using the same TiO2 nanowires as a matrix for LDI-TOF mass spectrometry. As the first step, the optimal condition for the photocatalytic decomposition of the peptides was determined, and the performance of the TiO2 nanowires synthesized using wet-corrosion was compared to that of a TiO2 nanoparticle (P25) solution. By using three model peptides with different number of amino acids, peptide sequencing was demonstrated with the TiO2 nanowires obtained from the wet-corrosion process. Additionally, the sequencing of three different peptides having the same molecular weights was demonstrated by using the TiO2 nanowires synthesized through wet-corrosion.

2. Materials and methods 2.1 Materials The Ti plates (1 mm thickness, >99.6% purity) used were purchased from Goodfellow Ltd. (Cambridge, England). TiO2 nanoparticles (P25) were purchased from Evonik Industries AG (Essen, Germany). KOH, potassium ferricyanide (K3[Fe(CN)6]), and methylene blue were purchased from Sigma-Aldrich (St. Louis, MO, US). The MSP 96 target plate for MicroScout MALDI-TOF mass spectroscopy was purchased from Bruker Daltonics Co. (Bremen, Germany). Six types of peptides (GHP9, PreS1, BPA-1, HPQ peptide-1, HPQ peptide-2, and HPQ peptide-3) were purchased from Peptron (Daejeon, Korea). Deionized water was obtained from a Milli-Q water purification system (Millipore Co; Bedford, UK).

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2.2 Synthesis of TiO2 nanowires TiO2 nanowires were synthesized by a modified wet-corrosion process:27-29, 45-51 (1) KOH treatment (TA), (2) water treatment (TAW), and (3) heat treatment (TAWH). As shown in Fig. 1(a), the etching process was performed by dipping the Ti plates in a 10 M KOH solution (Duksan Chemical Co., Korea) at room temperature for 24 h with mild shaking (TA). After the KOH treatment, the Ti plates were rinsed with distilled water and then dipped in the water at room temperature for 48 h with mild agitation (TAW). Finally, the Ti plates were thoroughly washed with distilled water and heat treated at 600 °C for 2 h (TAWH).

2.3 Characterization of TiO2 nanowires The change in the surface morphology of the Ti plate during the wet-corrosion process was observed using field-emission scanning electron microscopy (FE-SEM) with a microscope purchased from JEOL Co. (Japan). As shown in Fig. 1(b), the TiO2 nanowires were observed to have a three-dimensional (3D) networked structure with an average diameter of 30 nm. The crystal structure of the synthesized TiO2 nanowires was characterized at each step of the wet-corrosion process using high-resolution X-ray diffraction (HR-XRD; X’pert PRO, PAN analytical Co., USA) with a Cu-Kα X-ray source at 40 kV and 40 mA, a step rate of 2θ = 3°/min, and a glancing angle of θ = 1° with respect to the incident beam. As shown in Fig. 1(c), the XRD pattern of the Ti plate was observed after each step of the hydrothermal process. The XRD patterns after KOH (TA) and water treatments (TAW) showed the same peaks as the untreated Ti plate (Ti). As

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shown in Fig. 1(d), the change in the Ti surface state according to the wet-corrosion process was also analyzed by Raman spectroscopy (LabRam ARAMIS, Horiba Jobin-Yvon Co., France) with an Ar-ion laser at a wavelength of 514.5 nm and an incident laser power of 0.5 mW. As shown in Fig. 2(e), the changes in surface morphology before and after TiO2 nanowire synthesis by the wet-corrosion process were observed by using an atomic force microscope (AFM; X-100) obtained from Park System Co (Seoul, Korea).

2.4 Photocatalytic decomposition of peptides For the photocatalytic decomposition of the peptides, each peptide solution (0.5 μL) at the concentration of 1 mg/mL was dispensed on the TiO2 nanowires synthesized using the wet-corrosion process. The UV treatment for the photocatalytic decomposition of the peptides was performed using a UV lamp as part of a UV-lithography system at the radiation power of 23 mW/cm2 with a wavelength of 254 nm (from OS-1k exposure; Seoul, Korea). The TiO2 nanowire plate with the dropped sample was positioned at a distance of 75 cm from the UV lamp. The UV irradiation time was controlled to be 30, 60, and 120 s. The UV dose was calculated according to the treatment time in units of millijoules (or milliwatt-seconds) per square centimeter. The UV radiation power was additionally calibrated using a power meter (Newport 1918-C, Newport, CA, USA).52-53

2.5 LDI-TOF mass spectrometry with TiO2 nanowires LDI-TOF mass spectrometry was performed by bonding the TiO2 nanowire plate to a commercial steel target (MSP 96 target, ground steel, MicroScout Target) obtained from Bruker Daltonics Co. (Bremen, Germany). An electrical contact between the TiO2 nanowire

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plate and steel target was fabricated by using a Ag nanoparticle-containing paste. Each peptide sample was dropped onto the TiO2 nanowires, with a volume of 0.5 μL per sample spot. Mass spectrometric analysis was performed in a Microflex mass spectrometer obtained from Bruker Daltonics (Bremen, Germany) and equipped with a N2 laser (337 nm). The maximum laser energy was set to 70 μJ, with an offset of 40% and a laser pulse rate of 60 Hz. The mass spectrum was obtained in the positive ion mode with a first accelerating voltage of 20 kV and a second ion source voltage of 18 kV. The reflector voltage was set to 9.5 kV, and all the mass spectra were obtained by integrating 500 laser pulses for each sample.

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3. Results and discussion 3.1 Photocatalytic activity of TiO2 nanowires synthesized from wet-corrosion process TiO2 nanowires were synthesized by wet-corrosion of the Ti plate. As shown in Fig. 1(a), wet corrosion of the Ti plate was performed in three synthesis steps: (1) alkali treatment of the Ti plate in 10 M KOH for 24 h, (2) water treatment for 48 h to eliminate the alkali solution, and (3) heat treatment at 600 ℃ for 2 h. The change in the surface morphology was observed by SEM, and a porous structure was observed on the Ti plate after alkali treatment as shown in Fig. 1(b). The wet-corrosion process of Ti was reported to be the reaction of Ti with hydroxide ion to form TiO2 as follows:54 Ti + 3OH- → Ti(OH)3+ + 4eTi(OH)3+ + e- → TiO2∙H2O + 1/2H2 The formed TIO2 reacted again with hydroxide ion to form TIO2 as follows: 2TIO2 + 4OH- → H2Ti2O4(OH)2 If the concentration of K+ ion was high in the solution, a portion of H2Ti2O4(OH)2 reacted with K+ ion as follows: 2K+ + H2Ti2O4(OH)2 → K2Ti2O4(OH)2 + 2H+ Therefore, H2Ti2O4(OH)2 and K2Ti2O4(OH)2 were produced together, and exhibited different crystal structures of anatase and lepidocrocite-type layered structures based on the size difference of K+ ion and proton, respectively.

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The crystal structure of TiO2 nanowires were observed by XRD at each step. As shown in Fig. 1(c), in addition to strong Ti metal-related XRD peaks at high angle region, weak Bragg reflections were discernible at 2Θ = ~11.1° and ~24.x° for the TAWH material, which correspond to (020) reflection of layered titanate and (101) peak of anatase TiO2, respectively.55 This result strongly suggested the formation of layered titanate and anatase TiO2 phases by wet-corrosion process. However, it was hard to specify the structure-type of layered titanate component in TAWH from several layered titanate phases like trititanate-type and lepidocrocite-type layered titanate, since the basal spacings of layered titanate phases were strongly dependent not only on the type of titanate layer but also on the amount of interlayer cations and water molecules. The weak intensity of titanium oxide-related XRD peaks reflected the poor long-range crystal order of titanium oxides in TAWH. Thus, micro-Raman spectroscopic analysis was also carried out to determine the crystal structure of titanium oxide, since this spectroscopy could provide a sensitive probe to local crystal structure of poorly crystalline metal oxide. As shown in Fig. 1(d), the TAWH material exhibited several phonon lines corresponding to lepidocrocite-type titanate phase at 280, 450, and 680 cm-1 as well as strong Raman peaks of anatase TiO2 phase.55 These spectral features were obviously distinguishable from complex Raman features of trititanate-type layered titanate in the range of 100 – 300 cm-1.56 From these results of XRD and Raman spectroscopy, the synthesized TiO2 nanowires were determined to be crystallized with mixed phases of anatase TiO2 and lepidocrocite-type layered titanate. The microstructures of the Ti plate before and after the wet-corrosion process were also observed using atomic force microscopy (AFM). As shown in Fig. 1(e), the surface

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roughness (Rq) was observed to have similar values of 185.8 and 184.0 nm before and after the wet-corrosion process for TiO2 nanowire synthesis, respectively. However, the nanowire structure was observed to have a homogeneous distribution on the surface. A reaction with methylene blue was used to estimate the photocatalytic activity of the synthesized TiO2 nanowires. The photocatalytic activity of TiO2 nanostructures oxidized methylene blue, and the blue color of the dye became transparent when exposed to UV radiation of 365 nm as shown in Fig. 2(a). The relative photocatalytic activity of the synthesized TiO2 nanowires on a Ti plate was compared with a commercial TiO2 nanoparticle (P25). The TiO2 nanoparticle (P25) solutions (reaction volume of 0.1 ml) and the synthesized TiO2 nanowires on a Ti plate of an area 130 mm2 were used for the photocatalytic oxidation of methylene blue. During the UV radiation, the optical density (OD) was measured at the wavelength of 650 nm at every 10 min. As shown in Fig. 2(b), TiO2 nanoparticle (P25) solution at a concentration of 100 μg/ml (at the reaction volume of 0.1 ml including 10 μg of TiO2 nanoparticles) showed approximately the same photocatalytic activity as the synthesized TiO2 nanowires on the Ti plate at an area of 130 mm2. From this calculation, the catalytic activity of the synthesized TiO2 nanowires on the Ti plate at the unit area (1 mm2) was correlated to the amount of TiO2 nanoparticles (P25) of 77 ng. Usually, the difference in crystal structures of TiO2 nanostructures were known to influence photocatalytic activity. For example, TiO2 nanoparticles called P25 were known to have mixed crystal structures of anatase and rutile in the proportion of 3:1,56 and TiO2 nanoparticles (P25) were known to have a higher photocatalytic activity than a pure anatase or a pure rutile structure. As previously mentioned, the synthesized TiO2

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nanowires had mixed crystal structures of anatase and lepidocrocite-type layered structures, and they had a different crystal structure from that of TiO2 nanoparticle (P25), although both nanostructures had a comparable photocatalytic activity as shown in Fig. 2(b). The UV radiation time was optimized for the photocatalytic decomposition of peptides with the synthesized TiO2 nanowires. First, the peptide sample dropped on the synthesized TiO2 nanowires were exposed to UV radiation, and the peptide fragments were analyzed by LDI-TOF mass spectrometry. As shown in Fig. 2(c), three different UV radiation times of 30 s, 60 s, and 120 s were applied to the GHP9 peptide and the fragmented peptides were analyzed using the same synthesized TiO2 nanowires as a matrix for LDI-TOF mass spectrometry. Before UV radiation, the synthesized TiO2 nanowires showed no background mass peak, and only the mass peak of the mother peptide [M+H2O+Na]+= 1047.23 was observed from the peptide sample. After UV radiation the intensity of the mother peak decreased significantly, and the number and intensity of the mass peaks of the fragmented peptide were observed to have increased. In this work, the optimal UV exposure time was determined to be 30 s to obtain the maximum number of peptide fragments. In this work, the photocatalytic activity of the TiO2 nanostructures was used to analyze amino acid sequences of peptides. As a first step, the performance of the synthesized TiO2 nanowires for photocatalytic decomposition of peptides was compared with TiO2 nanoparticles (P25). When the GHP9 peptide with nine amino acids in the sequence G-HP-Q-G-K-K-K-K (Mw: 1006.59 Da) was photocatalytically decomposed by the synthesized TiO2 nanowires and the TiO2 nanoparticles (P25), the GHP9 peptide was decomposed

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into different peptide fragments as shown in Fig. 2(d). The synthesized TiO2 nanowires resulted in eleven peptide fragments including the mother peak of [GHP9+H2O+Na]+= 1047.23, and TiO2 nanoparticles (P25) produced three peptide fragments including the mother peak of [GHP9+K]+= 1045.41. These results showed that the synthesized TiO2 nanowires could produce a larger number of peptide fragments in comparison with TiO2 nanoparticles (P25). These results were attributed to the structural differences of the two TiO2 nanostructures. As the synthesized TiO2 nanowires had a more porous structure than TiO2 nanoparticles (P25), peptide molecules could be in contact with the TiO2 surface of the synthesized nanowires for a longer time than the TiO2 nanoparticles (P25). In addition, direct exposure to laser radiation was more easily avoided for peptide molecules treated with the synthesized TiO2 nanowires than for those treated with the TiO2 nanoparticles (P25). These results showed that the structural differences of the synthesized TiO2 nanowires and the TiO2 nanoparticles (P25) promoted the production of a larger number of peptide fragments.

3.2 Photocatalytic decomposition of peptides for amino acid sequencing This study analyzed the amino acid sequence of a specific peptide by the following steps: (1) photocatalytic decomposition of a peptide with the synthesized TiO2 nanowires and (2) LDI-TOF mass spectrometry with the synthesized TiO2 nanowires as the solid matrix. Therefore, peptide fragmentation as well as LDI-TOF mass spectrometry were performed on the same TiO2 nanowires. In principle, peptide fragmentation occurs at different breakage positions on a peptide bond, and the fragments can be assigned values such as a, b, and c (fragments of the N-terminal of peptides) and x, y, and z

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(fragments of the C-terminal of peptides), as shown in Fig. 3. In this study, the photocatalytic decomposition of peptide was performed at several sample spots, which were independently selected on the synthesized TiO2 nanowires using the same peptide sample. Usually, different sample spots are considered to form different peptide fragments. First, the GHP9 peptide with nine residues of amino acids in the sequence G1-H-P-QG2-K1-K2-K3-K4 (Mw: 1006.59 Da) was analyzed by photocatalytic decomposition with the synthesized TiO2 nanowires. In this peptide, two types of amino acids occurred multiple times (four lysine residues and two glycine residues). The mother peak of GHP9 was observed as [GHP9+H2O+Na]+ = 1047.23, as shown in Fig. 4(b). After the photocatalytic decomposition with the synthesized TiO2 nanowires, mass peaks of the peptide fragments appeared in an m/z range lower than that of the mother peak of GHP9. As shown in Fig. 4(c), the peptide fragments appeared as products of the photocatalytic decomposition with the synthesized TiO2 nanowires. When the amino acid sequence of the GHP9 peptide was analyzed based on the peptide fragments obtained after photocatalytic decomposition, the mass peaks of the peptide fragments at a single sample spot were determined to be insufficient for complete sequencing. Therefore, mass peaks at three different sample spots were collected and assigned for the analysis of the complete amino acid sequence of the GHP9 peptide, as shown in Fig. 4(d). As summarized in Table 1, each mass peak was assigned to a peptide fragment formed due to peptide bond breakage within the GHP9 peptide to achieve the complete amino acid sequencing. These results showed that the complete amino acid sequence of the GHP9

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peptide could be analyzed using the mass peaks of its photocatalytic decomposition with the synthesized TiO2 nanowires. BPA-1 peptide with 13 amino acid residues in the sequence K1-S1-L-E-N-S2-Y-G1-G2-G3K2-K3-K4 (Mw: 1394.74 Da) was also analyzed. In this peptide, three types of amino acids occurred multiple times (four lysine residues, two serine residues, and three glycine residues). The mother peak of BPA-1 was observed as [BPA-1+Na]+ = 1417.50 in Fig. 5(b). After photocatalytic decomposition on the synthesized TiO2 nanowires, mass peaks of the BPA-1 peptide appeared in an m/z range lower than that of the mother peak of BPA-1. As shown in Fig. 5(c), peptide fragments were observed as products of the photocatalytic decomposition with the synthesized TiO2 nanowires. When the amino acid sequence of the BPA-1 peptide was analyzed based on the peptide fragments obtained by photocatalytic decomposition, the mass peaks of the peptide fragments at a single sample spot were insufficient for complete sequencing. Therefore, mass peaks at three different sample spots were collected and assigned for the analysis of the complete amino acid sequence of the BPA-1 peptide, as shown in Fig. 5(d). As summarized in Table 2, each mass peak was assigned to a peptide fragment formed due to peptide bond breakage within the BPA-1 peptide to achieve the complete amino acid sequencing. These results showed that the complete amino acid sequence of the BPA-1 peptide could be analyzed using the mass peaks of its photocatalytic decomposition with the synthesized TiO2 nanowires. PreS1 peptide with 15 amino acid residues in the sequence F1-G-A-N1-S-N2-N3-P1-D1W-D2-F2-N4-P2-N5

(Mw:

1707.68

Da)

was

also

analyzed

through

photocatalytic

decomposition with the synthesized TiO2 nanowires. In this peptide, four types of amino

15



acids were observed multiple times (two phenylalanine residues, two proline residues, two aspartate residues, and five asparagine residues). The mother peak of PreS1 was observed as [PreS1-H2O+K]+ = 1728.63, as shown in Fig. 6(b). After photocatalytic decomposition with the synthesized TiO2 nanowires, mass peaks from the PreS1 peptide appeared in an m/z range lower than that of the mother peak of PreS1. As shown in Fig. 6(c), the peptide fragments were observed as products of the photocatalytic decomposition with the synthesized TiO2 nanowires. When the amino acid sequence of the PreS1 peptide was analyzed based on the peptide fragments formed by photocatalytic decomposition, the mass peaks of peptide fragments at a single sample spot were sufficient for complete sequencing of PreS1, as shown in Fig. 6(d). As summarized in Table 3, each mass peak was assigned to a peptide fragment formed due to peptide bond breakage within the PreS1 peptide to achieve the complete amino acid sequencing. These results showed that the complete amino acid sequence of the PreS1 peptide could be analyzed using the mass peaks of its photocatalytic decomposition with the synthesized TiO2 nanowires.

From the analysis results of each peptide the selectivity on the photocatalytic decomposition of the amide bond was hardly found from the mass peaks of six types of peptide. As shown in Tables, each mass peak from the breakage for peptide bonds, such as a, b, c (from N-terminal) and x, y, z (from C-terminal) was nearly observed only a single time even when more than two independently prepared sample spots were applied to the photocatalytic decomposition with the synthesized TiO2 nanowires. These results showed that the reproducibility to 16


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produce the same peptide fragment were very low even when the photocatalytic decomposition was carried out for the same peptides. Additionally, the binding affinity of the peptide fragments to the Na+, K+ and proton was observed to be randomly occurred as shown in Tables for the assignment of each mass peaks.

3.3 Photocatalytic decomposition of three different peptides with the same molecular weights As shown in the analysis of the three peptides (GHP9, BPA-1, PreS1), the photocatalytic decomposition with the synthesized TiO2 nanowires could form many peptide fragments. Here, peptides with different amino acid sequences and the same molecular weights have been analyzed using photocatalytic decomposition with the synthesized TiO2 nanowires. As shown in Fig. 7(a), the amino acid compositions of the three peptides were designed to have the same molecular weight of 884.45 Da. The mass spectrum of each peptide showed the same m/z ratio of 885.26, and it was very difficult to distinguish these peptides without tandem mass spectrometry. As shown in Fig. 7(b), the peptide fragments were observed as products of the photocatalytic decomposition with the synthesized TiO2 nanowires. When the amino acid sequence of the three HPQ peptides was analyzed based on the peptide fragments formed by photocatalytic decomposition, mass peaks of peptide fragments at a single sample spot were determined to be insufficient for complete sequencing. For HPQ peptide-1, the mass peaks at three different sample spots were collected and analyzed together, while HPQ peptide-2 and -3 could be completely sequenced by collecting mass peaks at two sample spots, as shown in Fig. 7(c). As summarized in Table 4, each mass

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peak was assigned to a peptide fragment formed due to peptide bond breakage within each HPQ peptide to achieve the complete amino acid sequencing of the three HPQ peptides.

4. Conclusions TiO2 nanowires were synthesized from a Ti plate through a wet-corrosion process. The microstructural changes during the wet-corrosion process were observed by SEM and AFM. The results of XRD analysis and Raman spectroscopy show that the synthesized TiO2 nanowires were be titanate with a mixed crystal structure of anatase and lepidocrocite-type

layered

structures.

The

relative photocatalytic activity

of the

synthesized TiO2 nanowires on a Ti plate was compared with that of a commercial TiO2 nanoparticle (P25) solution. Further, the catalytic activity of the synthesized TiO2 nanowires on Ti plate at the unit area (1 mm2) was correlated to the amount of TiO2 nanoparticle (P25) solution of 77 ng. Additionally, the optimal UV radiation time was determined to be 30 s for the photocatalytic decomposition of peptides with the synthesized TiO2 nanowires. The feasibility of peptide sequencing using the synthesized TiO2 nanowires was demonstrated for the following model peptides: (1) GHP9, which contains nine residues in the sequence of G1-H-P-Q-G2-K1-K2-K3-K4 (Mw: 1006.59 Da); (2) BPA-1, which has 13 residues in the sequence of K1-S1-L-E-N-S2-Y-G1-G2-G3-K2-K3-K4 (Mw: 1394.74 Da), and (3) PreS1, which comprises 15 residues in the sequence of F1-G-AN1-S-N2-N-P1-D1-W-D2-F2-N3-P-N4 (Mw: 1707.68 Da). The mass peaks of the peptide fragments from a single sample spot were insufficient for the complete sequencing of the peptides. In this study, mass peaks from three different sample spots were collected

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and used for the analysis of the complete amino acid sequence of each peptide. Subsequently, three peptides with different amino acid sequences but the same molecular weight were analyzed via photocatalytic decomposition with the synthesized TiO2 nanowires.

ACKNOWLEDGMENT The authors thank to Prof. Seong-Ju Hwang and Dr. Jo-Il Kim for valuable contribution to the revision of this paper. This work was supported by the Nano-Convergence Foundation [grant number: R201602210] funded by the Ministry of Science, ICT and Future Planning (MSIP, Korea) and the Ministry of Trade, Industry and Energy (MOTIE, Korea), by the Industry Technology Development Program [grant number: 10063335] funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea), and by the National Research Foundation of Korea [grant number: NRF-2017R1A2B4004077, NRF2017R1A2B2004398].

19



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ACS Applied Materials & Interfaces 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

Table of contents/Abstract graphic

24


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Figure legends

Figure 1. TiO2 nanowires synthesized through the wet-corrosion process. (a) Wet-corrosion process of the Ti plate for the synthesis of TiO2 nanowires. (b) SEM images of the Ti plate after each step of the wet-corrosion process. TA denotes the Ti plate surface after alkali treatment, TAW the surface after alkali and water treatments, and TAWH the surface after alkali, water, and heat-treatments. (c) XRD patterns of the Ti plate after each step of the wet-corrosion process. (d) Raman spectra of the Ti plate after each step of the wetcorrosion process. (e) AFM images of the Ti plate before and after the wet-corrosion process.

Figure 2. Photocatalytic reactivity of the TiO2 nanowires and TiO2 nanoparticle (P25) solution. (a) Principle of the photocatalytic reaction of anatase TiO2. (b) Comparison of the photocatalytic activities of the TiO2 nanowires and TiO2 nanoparticle (P25) solutions at different concentrations. (c) Optimization of UV radiation time for photocatalytic decomposition of the synthesized TiO2 nanowires. (d) Comparison of the photocatalytic decomposition pattern of GHP9 peptide with that of the synthesized TiO2 nanowires and TiO2 nanoparticle (P25).

Figure 3. Peptide fragment assignment according to the breakage position of the peptide bond. The fragment assignments were noted as a, b, c (from N-terminal) and x, y, and z (from C-terminal).

25



Figure 4. Peptide sequencing of the GHP9 peptide. (a) Fragment analysis of the GHP9 peptide. (b) LDI mass spectra of the GHP9 peptide before and after photocatalytic decomposition. (c) Fragment analysis of the GHP9 peptide from the LDI mass spectra. (d) Peptide sequencing from the mass peaks of the photocatalytic decomposition of the GHP9 peptide.

Figure 5. Peptide sequencing of the BPA-1 peptide. (a) Fragment analysis of the BPA peptide. (b) LDI mass spectra of the BPA-1 peptide before and after photocatalytic decomposition. (c) Fragment analysis of the BPA-1 peptide from the LDI mass spectra. (d) Peptide sequencing from the mass peaks of the photocatalytic decomposition of the BPA-1 peptide.

Figure 6. Peptide sequencing of the PreS1 peptide. (a) Fragment analysis of the PreS1 peptide. (b) LDI mass spectra of the PreS1 peptide before and after photocatalytic decomposition. (c) Fragment analysis of the PreS1 peptide from the LDI mass spectra. (d) Peptide sequencing from the mass peaks of the photocatalytic decomposition of the PreS1 peptide.

Figure 7. Peptide analysis of the three different peptides with the same molecular weights (884.45 Da). (a) Fragment analysis of the three peptides. (b) Peptide sequencing from the mass peaks of the photocatalytic decomposition of the three peptides.

26


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Figure 1

(a)

27



(b)

28


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(c)

(d)

29



(e)

30


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Figure 2

(a)

31



(b)

32


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(c)

33



(d)

34


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Figure 3

35



Figure 4 (a)

(b)

(c)

36


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(d)

37



Page 38 of 49

Table 1. Amino acid sequencing of the GHP9 peptide by the mass peaks from the photocatalytic reaction with the synthesized TiO2 nanowires. Sequence GHPQGKKKK GHPQGKKK GHPQGKK GHPQGK GHPQG

Observed m/z Monoisotropic 1045.12 869.03 707.42 587.89 516.80

Expected Ion Form* [GHPQGKKKK-H+K]+ +

[(GHPQGKKK-OH)-NH+Na] +

[(GHPQGKK-COO)+H]

+ㆍ

[(GHPQGK-OH)-NH2-H]

+

[(GHPQG-OH+NH3)-H+Na] +ㆍ

Calculated m/z

Fragment

Monoisotropic

Assign

Spectrum #

1045.59

y9

3

868.50

b8

1

707.43

a7

3

588.29

b6

2

516.23

c5

2

GHPQ

418.59

[(GHPQ-OH)-H]

419.19

b4

3

GHP

324.53

[(GHP-OH+NH3)-H+NH3]+

325.19

c3

1

GH

211.38

[(GH-OH+NH3)]+

212.11

c2

1

97.01

-

2

G

96.79

+

[G-H+Na]

* Expected ion form of peptide fragment from the actually measured mass peak. “Spectrum #” meant the number of mass spectrum with the actually measured mass peak.

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Figure 5 (a)

(b)

39



(c)

40


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(d)

41



Page 42 of 49

Table 2. Amino acid sequencing of the BPA-1 peptide by the mass peaks from the photocatalytic reaction with the synthesized TiO2 nanowires. Sequence KSLENSYGGGKKK KSLENSYGGGKK KSLENSYGGGK KSLENSYGGG KSLENSYGG KSLENSYG

Observed m/z Monoisotropic 1414.47 1304.99 1122.54 1012.12 940.92 893.92

Expected Ion Form* [(KSLENSYGGGKKK-OH)-H+K]+ +

[(KSLENSYGGGKK-OH+NH3)-H+K] +

[(KSLENSYGGGK-OH)+H]

+

[(KSLENSYGGG-OH+NH3)-2H2O-H+K] +

[(KSLENSYGG-OH)-H2O+Na] +ㆍ

[(KSLENSYG-OH)+NH]

+

Calculated m/z

Fragment

Monoisotropic

Assign

Spectrum #

1415.71

b13

3

1304.64

c12

2

1122.55

b11

3

1012.43

c10

2

941.42

b9

3

894.41

b8

1

KSLENSY

802.60

[(KSLENSY-COOH)-NH+Na]

802.39

a7

2

KSLENS

678.53

[(KSLENS-OH)-H2O-H+K]+

679.28

b6

3

577.28

b5

3

497.27

c4

2

KSLEN KSLE KSL KS K

577.45 496.62

+

[(KSLEN-OH)-H2O+Na]

+

[(KSLE-OH+NH3)-H+Na] +

384.38

[(KSL-OH+NH3)-H+K]

384.21

c3

2,3

271.21

+

271.12

c2

3

185.10

-

3

185.00

[(KS-OH+NH3)-H+K] +

[K+K]


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Figure 6 (a)

(b)

43



(c)

44


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(d)

45



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Table 3. Amino acid sequencing of the PreS1 peptide by the mass peaks from the photocatalytic reaction with the synthesized TiO2 nanowires Sequence FGANSNNPDWDFNPN FGANSNNPDWDFNP FGANSNNPDWDFN FGANSNNPDWDF

Observed m/z Monoisotropic 1692.84 1558.03

Expected Ion Form* [(FGANSNNPDWDFNPN-NH2)+H]+ +

[(FGANSNNPDWDFNP-OH)-H2O]

+

Calculated m/z

Fragment

Monoisotropic

Assign

Spectrum #

1692.68

z15

1

1558.64

b14

1

1521.09

[(FGANSNNPDWDFN-OH)+H2O+Na]

1520.59

b13

1

1364.95

+

1364.57

c12

1

[(FGANSNNPDWDF-OH+NH3)-H2O] +

FGANSNNPDWD

1239.87

[(FGANSNNPDWD-OH)-H+Na]

1240.46

b11

1

FGANSNNPDW

1105.51

[(FGANSNNPDW-OH)+H]+

1104.46

b10

1

921.37

b9

1

FGANSNNPD FGANSNNP FGANSNN FGANSN FGANS

921.36

+

[(FGANSNNPD-OH)-H2O-H+Na] +

824.80

[(FGANSNNP-OH)-H+Na]

824.34

b8

1

729.27

+

729.29

b7

1

598.24

b6

1

475.22

c5

1

598.10 475.17

[(FGANSNN-OH)-NH+K]

+

[(FGANSN-OH)-NH2+Na]

+ㆍ

[(FGANS-OH+NH3)-H2O-H] +

FGAN

397.61

[(FGAN-OH)-NH2+Na]

397.17

b4

1

FGA

288.21

[(FGA-COOH)+H+K]+

288.13

a3

1

FG

243.05

[(FG-OH)-H+K]+

243.09

b2

1

159.05

a1

1

F

159.09

+

[(F-COOH)+K]


46


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Figure 7 (a)

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(b)

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Table 4. Amino acid sequencing of the three HPQ peptides by the mass peaks from the photocatalytic reaction with the synthesized TiO2 nanowires. Peptide

Sequence GYHPQRK

HPQ peptide-1 (GYHPQRK)

HPQ peptide-2 (KRHPQYG)

HPQ peptide-3 (RYHPQGK)

Observed m/z Monoisotropic 923.14

Expected Ion Form* [GYHPQRK+K]+ +

Calculated m/z

Fragment

Monoisotropic

Assign

Spectrum #

923.45

y7

2,3 2

GYHPQR

724.40

[(GYHPQR-OH)-NH2+H]

724.36

b6

GYHPQ

588.92

[(GYHPQ-OH)-NH2-H+Na]+

589.26

b5

1

GYHP

477.92

[(GYHP-OH)+Na]+

478.20

b4

1,2

GYH

374.02

[(GYH-OH+NH3)-H]+ㆍ

374.14

c3

3

GY

276.11

[(GY-OH+NH3)-H+K]+

276.08

c2

1,3

G

112.16

[(G-OH+NH3)-2H+K]+ㆍ

112.02

c1

1,2

KRHPQYG

889.82

[(KRHPQYG-OH)+Na]+

889.45

b7

1

KRHPQY

828.63

+

[(KRHPQY-OH+NH3)]

827.46

c6

1

KRHPQ

658.65

[(KRHPQ-COOH)+K]+

658.37

a5

1,2

KRHP

536.29

[(KRHP-OH+NH3)]+

536.31

c4

2

+ㆍ

KRH

377.96

[(KRH-COOH)-NH2]

378.26

a3

1

KR

297.25

[(KR-COOH)+H+K]+

297.18

a2

2

+

K

152.46

[(K-OH)+Na]

152.10

b1

1

RYHPQGK

907.04

[RYHPQGK+Na]+

907.45

y7

1

RYHPQG

795.73

[(RYHPQG-OH+NH3)+K]+

795.36

c6

2

RYHPQ

702.45

[(RYHPQ-OH)-H2O-H+K]+

702.29

b5

1

RYHP

526.90

[(RYHP-COOH)+H]+

527.28

a4

1

RYH

468.72

[(RYH-COOH)+K]+

468.22

a3

1

RY

294.25

[(RY-COO)+H]+

294.16

a2

1

175.10

-

2

R

175.37

+

[R+H]

* Expected ion form of peptide fragment from the actually measured mass peak. “Spectrum #” meant the number of mass spectrum with the actually measured mass peak. 49


TiO2 nanowires from wet-corrosion synthesis for peptide sequencing

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