Accepted Manuscript Title: Efficient Protein Digestion Using Immobilized Trypsin onto Tannin Modified Fe3 O4 Magnetic Nanoparticles Author: Keziban Atacan Bekir Cakiroglu Mahmut Ozacar PII: DOI: Reference:
S0927-7765(17)30252-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.04.055 COLSUB 8523
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10-1-2017 24-3-2017 27-4-2017
Please cite this article as: K. Atacan, B. Cakiroglu, M. Ozacar, Efficient Protein Digestion Using Immobilized Trypsin onto Tannin Modified Fe3 O4 Magnetic Nanoparticles, Colloids and Surfaces B: Biointerfaces (2017), http://dx.doi.org/10.1016/j.colsurfb.2017.04.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficient Protein Digestion Using Immobilized Trypsin onto Tannin Modified Fe3O4 Magnetic Nanoparticles Keziban Atacana, Bekir Cakiroglua, Mahmut Ozacara,b* a
Ac
ce pt
ed
M
an
us
cr
ip t
Biomedical, Magnetic and Semiconductor Materials Research Center (BIMAS-RC), Sakarya University, Sakarya 54187, Turkey b Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey
*
Corresponding Author. Tel.:+90 264 295 60 41; fax:+90 264 295 59 50. E-mail address: [email protected] (M.Ozacar).
Page 1 of 36
2
ce pt
ed
M
an
us
cr
ip t
Graphical Abstract
Ac
The modification and immobilization processes of Fe3O4 MNPs for efficient BSA digestion.
Page 2 of 36
3 Highlights Fe3O4 MNPs was synthesized by solvothermal method. Trypsin was immobilized onto tannin modified Fe3O4 MNPs. Decrease of digestion time up to 1 min. Immobilized trypsin exhibits high digestion capacity of BSA protein using MALDI-
ip t
TOF MS.
The BSA and egg white proteins were digested efficiently by observing with SDS-
Ac
ce pt
ed
M
an
us
cr
PAGE.
Page 3 of 36
4 Abstract: Fe3O4 magnetic nanoparticles (MNPs) were prepared via solvothermal method. A commercially available trypsin was covalently immobilized onto MNPs modified with tannin (T) via a novel binding process. The morphology, structure, surface and magnetic properties of the obtained nanostructures were characterized comprehensively. The Fe3O4 MNPs had a
ip t
saturation magnetization value of 60.18 emu/g at room temperature, while the tannin modified Fe3O4 MNPs, and the trypsin immobilized on tannin modified-Fe3O4 MNPs possessed a
cr
saturation magnetization values of 57.82 emu/g and 55.16 emu/g, respectively, which
us
indicated the decent tannin coating and trypsin immobilization. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of
an
widely used bovine serum albumin (BSA). The immobilized trypsin was investigated by conducting the tryptic digestion of BSA within 1 min, 5 min and 15 min. Also, microwave-
M
assisted digestion was carried out for 15 s. The digested protein fragments were identified by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF
ed
MS), and a satisfactory peptide numbers of 39, and a superior sequence coverage of 84% for 1 min digestion were obtained. The sodium dodecyl sulphate–polyacrylamide gel
ce pt
electrophoresis (SDS–PAGE) analysis confirmed the satisfactory digestion of BSA and egg white proteins by immobilized trypsin.
Keywords: Fe3O4, Covalent bonding, Trypsin immobilization, protein digestion, sequence
Ac
coverage.
Page 4 of 36
5
1. Introduction
Magnetic nanoparticles have been one of the most promising materials for biological
ip t
applications [1]. Especially Fe3O4 is preferable for drug delivery, biosensing [2] and other studies based on the enzyme immobilization over the other ones owing to its biocompatibility
cr
and superparamagnetic behavior [1]. Since the bare Fe3O4 nanoparticles often have poor stability and dispersity, various modification methods have been exploited to obtain soluble
an
have been extensively used for various applications [2].
us
and more biocompatible Fe3O4 nanoparticles. The resulting modified Fe3O4 nanoparticles
M
Surface modification methods have been developed to modify the surface of pristine Fe3O4 nanoparticles via loading of other chemicals or biological materials during or after the process
to
improve
the
dispersibility,
stability,
biocompatibility
and
ed
synthesis
biodegradability for specific purposes [3,4]. Tannic acid, a natural polyphenolic molecule
ce pt
consisting of acentral core of glucose linking to ten gallic acid groups throughester bonds, exists in a variety of plants [5,6]. The numerous hydroxyl groups can interact with structures and biopolymers suchas some proteins, digestive enzymes, carbohydrates, and minerals to
Ac
provide many intriguing physical and chemical properties of TA-based materials [7-9].
Immobilization of enzymes on solid supports can enhance enzyme stability and proteolysis efficiency as well as it prevents the enzyme contamination in the product and, enhances the recovery for reuse [10]. Along with the extraordinary development of nanotechnology, various nanostructures were explored as carrier material for immobilization of enzymes and improvement of respective activities [11–15]. Besides, the composition of the enzyme carriers
Page 5 of 36
6 plays an important role in biocompatibility, which has a pivotal role in keeping the enzyme structure unchanged by yielding enhanced activity and efficiency [16,17].
Proteases are widely used in industrial and biomedical applications, among which trypsin is
ip t
most extensively concerned. Trypsin is a pancreatic protease, and it cleaves peptide, ester, and amide bonds at the carboxylic side of arginine, lysine and ornithine residues. Of all the
us
catalyze hydrolysis of proteins in the biochemical reactions [18].
cr
digestive endopeptidases, trypsin has the most pronounced substrate specificity, and is used to
an
Protein digestion by using trypsin is of great importance in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and
M
nonautomated due to the poor stability and autolysis of trypsin [19]. Trypsin digestion has been one of the most widely used biological reactions for both research and industrial
ed
applications [20-22]. However, the rapid autolysis of trypsin in the solution leads to unwanted and interfering fragments and, consequently decreasing the efficiency and rate of catalytic
ce pt
reactions [23,24]. Therefore, trypsin immobilization can overcome these problems.
In our previous work [25], Fe3O4 MNPs were synthesized through the co-precipitation of Fe2+
Ac
and Fe3+ in an ammonia solution. Trypsin was immobilized on the tannic acid coated Fe3O4 MNPs via non-covalent immobilization. In addition, the influences of pH, temperature, thermal stability, storage time stability and reusability were studied. The results indicated that the activity of immobilized trypsin was predominantly preserved compared to the activity of free trypsin.
Page 6 of 36
7 Herein, Fe3O4 MNPs were prepared by solvothermal method and Fe3O4 MNPs were functionalized with tannin. Since the solvothermal method enable tailoring particle size and shape to fit appropriate applications, it can be preferable in nanoparticle synthesis [4,26]. Also, covalent immobilization of trypsin on tannin modified Fe3O4 MNPs was carried out by
ip t
the formation of Schiff-base reaction or Michael-type addition between the quinone groups on tannin modified nanoparticles, which result from the pH increment, and the amino groups of
cr
the trypsin. Finally, the prepared trypsin-linked Fe3O4 MNPs were successfully utilized for the
us
digestion of BSA. The illustration of the process was given in Scheme 1. The hydrolysis products of BSA and egg white proteins formed in the presence of immobilized trypsin were
procedures.
In
addition,
the
digests
an
analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were
identified
by
matrix-assisted
laser
M
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we aimed to produce facile synthesized, non-toxically modified, and strongly trypsin
ed
immobilized magnetic nanoparticles, which exhibit a high efficiency for the protein digestion. The fragments of BSA hydrolsed by immobilized trypsin were determined and identified via
ce pt
MALDI-TOF MS.
Ac
2. Materials and methods
2.1. Materials
Ferric chloride hexahydrate (FeCl3·6H2O, >99%), sodium acetate, di-sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium dodecyl sulphate, Folin-Ciocalteu’s phenol reagent, sodium hydroxide (≥97%), ethanol (>99.2%) were procured from Merck (Germany). Trypsin from bovine pancreas, Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA, 98%), albumin from bovine serum (BSA; 98%, agarose gel electrophoresis),
Page 7 of 36
8 Bradford Reagent, benzaimidine, 4-nitroaniline (p–nitroaniline), Tetramethylethylenediamine (TEMED), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), β-merkaptoetanol (99.0%), Tris(hydroxymethyl)aminomethane, acetic acid, methanol (>99.8%) were purchased from Sigma-Aldrich (USA). TGX Stain-FreeTM FastCastTM Acrylamide Starter Kit, 10% was
Protein
Ladder
(Protein
Gel
Electrophoresis)
was
ip t
purchased from Bio-Rad Laboratories (USA). PageRuler™ Unstained Low Range obtained
from
Thermo
cr
Scientific™ (USA). Human Serum sample was obtained from Sakarya University Medico-
us
Social Center. Ethylene glycol (EG) (99%, C2H6O2) and ammonium hydrogen carbonate were provided from Tekkim (Turkey). Deionized water (DW, with resistance >18MΩ/cm) was
an
produced by using Millipore Milli Q Plus purification system, and was used throughout this
M
work.
ed
2.2. Synthesis of Fe3O4 nanoparticles and functionalization with tannin
The magnetic Fe3O4 MNPs were prepared according to a reported solvothermal method
ce pt
[27,28]. Typically, FeCl3· 6H2O (4.440 g) and NaAc (14.40 g) were first dissolved in ethylene glycol (160 mL) and the mixture was stirred at 200 rpm for 1 h. Afterward, the solution was transferred to a 50 mL Teflon-lined stainless-steel autoclave. The autoclave was sealed and
Ac
heated at 200°C and maintained for 8 h. Then, it was allowed to cool to room temperature. The magnetic Fe3O4 MNPs were washed with ethanol and deionized water several times and separated by magnetic decantation, and washed with deionized water and ethanol thrice. Finally, the product was dried in vacuum at 70 °C for 12 h.
For the preparation of tannin coated Fe3O4 MNPs (Fe3O4–T), the as-synthesized Fe3O4 MNPs (2.9708 g) were dispersed in deionized water (60 mL) by ultrasonication for 15 min and
Page 8 of 36
9 mixed at 200 rpm under N2 atmosphere at 40 °C for 1 h. Tannin (1.4862 g) was dissolved in 40 mL of deionized water and was added into the Fe3O4 nanoparticles dispersion. The mixture was stirred at 200 rpm under N2 atmosphere at 40 °C for 2 h. Then, tannin coated Fe3O4 MNPs were separated using a neodymium magnet, and washed with deionized water and
ip t
ethanol thrice, after which the product was dried in vacuum at 70 °C for 12 h [25].
us
cr
2.3. Trypsin immobilization
0.5 g of as-prepared tannin coated Fe3O4 MNPs were subjected to 3 mL of sodium hydroxide
an
(0.1 M, pH 9.4) to impart the quinone group on the tannin coated nanoparticles by using the pH dependent oxidation reaction of polyphenols [29]. The resulting solution was stirred at
M
200 rpm for 1 h at room temperature. Then, the product was separated magnetically. Subsequently, 10 mg/mL trypsin solution (containing 6 mg/mL benzamidine in 0.1 M, pH 7.5
ed
sodium phosphate buffer solution, PBS) was added into base treated modified Fe3O4 MNPs. The mixture solution was stirred at 200 rpm at 4 °C for 3 h. The product was then separated
ce pt
magnetically, and was washed with the same buffer solution for three times. The resulting enzyme immobilized Fe3O4–T (Fe3O4–T–TR) was stored at 4 ◦C until use.
Ac
2.4. Digestion of BSA and Human Serum
Standard stock BSA solution (2 mg/mL) was prepared by dissolving 20 mg BSA in 10 mL NH4HCO3 solution (100 mM, pH 8). Prior to the digestion, BSA was denatured in a 95 °C water bath for 15 min to increase efficiency of protein cleaving [30,31]. 250 µL of stock solution of BSA and 750 µL NH4HCO3 solution (100 mM, pH 8) were mixed to obtain a BSA concentration of 0.5 mg/mL.
Page 9 of 36
10 10 mg immobilized trypsin was transferred into a 1.5 mL Eppendorf tube and then 100 µL of denaturated, and diluted BSA solution was added to the same tube (100 µg/µL). Then the mixture solution was incubated at 37 °C for 1 min, 5 min and 15 min, followed by the magnetically separation using a neodymium magnet. The liquid phase was transferred to an
ip t
Eppendorf tube for MALDI-TOF MS analysis. For comparison, 50 µL solution of denaturated BSA was transferred into an Eppendorf tube, and 10 µL trypsin solution containing 6 mg/mL
cr
benzamidine in NH4HCO3 solution (0.1 M, pH 8.0) with a concentration of 5 mg/10 mL was
us
added to the same tube. After the mixture was incubated in a 37 °C for 18 hours, digestion was quenched by the addition of 11 µL of glacial acetic acid. Then the mixture was examined
M
investigated according to the same method.
an
by MALDI-TOF MS. Also, human serum (6.7 mg/dL determined by Biuret method) was
As for the microwave-assisted BSA digestion, the similar method was conducted for free and
ed
immobilized trypsin except that after microwave irradiation (CEM Mars model) with a controlled power of 700 W for 15 s, the mixture solution was separated magnetically [32] and
ce pt
the liquid phase was transferred to an Eppendorf tube for MALDI-TOF MS.
Ac
2.5. MALDI-TOF MS procedure and data analysis
MALDI mass spectra were acquired by a Voyager-DE Pro MALDI-TOF mass spectrometer (Applied Biosystems, USA). Sample desorption and ionization was carried out using the 337 nm output from a pulsed nitrogen laser (Spectra Physics, USA) at ca. 2.0×10−7 Torr vacuum and acceleration voltage of 25 kV. All spectra were acquired by averaging between 100 laser shots. 10 mg of α-cyano-4-hydroxycinnamic acid and sinapinic acid were separately dissolved in 1 mL of CH3CN:water (3:1, v/v) mixture containing 0.1% (v/v) trifluoroacetic acid.
Page 10 of 36
11 Analyte solutions were mixed with matrix in 1:10 ratio (v/v) and 1 μL of this final mixture was directly spotted onto the MALDI plate. The analysis of the digested BSA samples was acquired using sinapinic acid matrix after air-drying of each spot [33]. The mascot MS/MS ions search (Mascot database search (www.matrixscience.com) parameters were as follows:
ip t
trypsin chosen as enzyme, swissprot chosen as database, peptide mass tolerance was set to ±0.4 Da and the MS/MS tolerance was set to ±0.2 Da, seven miscleavage of trypsin enzyme
cr
specificity was allowed, peptide charge state was set to +2, +3, and +4 and carbamidomethyl
us
was set as a variable modification. Peptide matches with significant homology (p
S0927-7765(17)30252-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.04.055 COLSUB 8523
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10-1-2017 24-3-2017 27-4-2017
Please cite this article as: K. Atacan, B. Cakiroglu, M. Ozacar, Efficient Protein Digestion Using Immobilized Trypsin onto Tannin Modified Fe3 O4 Magnetic Nanoparticles, Colloids and Surfaces B: Biointerfaces (2017), http://dx.doi.org/10.1016/j.colsurfb.2017.04.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficient Protein Digestion Using Immobilized Trypsin onto Tannin Modified Fe3O4 Magnetic Nanoparticles Keziban Atacana, Bekir Cakiroglua, Mahmut Ozacara,b* a
Ac
ce pt
ed
M
an
us
cr
ip t
Biomedical, Magnetic and Semiconductor Materials Research Center (BIMAS-RC), Sakarya University, Sakarya 54187, Turkey b Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey
*
Corresponding Author. Tel.:+90 264 295 60 41; fax:+90 264 295 59 50. E-mail address: [email protected] (M.Ozacar).
Page 1 of 36
2
ce pt
ed
M
an
us
cr
ip t
Graphical Abstract
Ac
The modification and immobilization processes of Fe3O4 MNPs for efficient BSA digestion.
Page 2 of 36
3 Highlights Fe3O4 MNPs was synthesized by solvothermal method. Trypsin was immobilized onto tannin modified Fe3O4 MNPs. Decrease of digestion time up to 1 min. Immobilized trypsin exhibits high digestion capacity of BSA protein using MALDI-
ip t
TOF MS.
The BSA and egg white proteins were digested efficiently by observing with SDS-
Ac
ce pt
ed
M
an
us
cr
PAGE.
Page 3 of 36
4 Abstract: Fe3O4 magnetic nanoparticles (MNPs) were prepared via solvothermal method. A commercially available trypsin was covalently immobilized onto MNPs modified with tannin (T) via a novel binding process. The morphology, structure, surface and magnetic properties of the obtained nanostructures were characterized comprehensively. The Fe3O4 MNPs had a
ip t
saturation magnetization value of 60.18 emu/g at room temperature, while the tannin modified Fe3O4 MNPs, and the trypsin immobilized on tannin modified-Fe3O4 MNPs possessed a
cr
saturation magnetization values of 57.82 emu/g and 55.16 emu/g, respectively, which
us
indicated the decent tannin coating and trypsin immobilization. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of
an
widely used bovine serum albumin (BSA). The immobilized trypsin was investigated by conducting the tryptic digestion of BSA within 1 min, 5 min and 15 min. Also, microwave-
M
assisted digestion was carried out for 15 s. The digested protein fragments were identified by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF
ed
MS), and a satisfactory peptide numbers of 39, and a superior sequence coverage of 84% for 1 min digestion were obtained. The sodium dodecyl sulphate–polyacrylamide gel
ce pt
electrophoresis (SDS–PAGE) analysis confirmed the satisfactory digestion of BSA and egg white proteins by immobilized trypsin.
Keywords: Fe3O4, Covalent bonding, Trypsin immobilization, protein digestion, sequence
Ac
coverage.
Page 4 of 36
5
1. Introduction
Magnetic nanoparticles have been one of the most promising materials for biological
ip t
applications [1]. Especially Fe3O4 is preferable for drug delivery, biosensing [2] and other studies based on the enzyme immobilization over the other ones owing to its biocompatibility
cr
and superparamagnetic behavior [1]. Since the bare Fe3O4 nanoparticles often have poor stability and dispersity, various modification methods have been exploited to obtain soluble
an
have been extensively used for various applications [2].
us
and more biocompatible Fe3O4 nanoparticles. The resulting modified Fe3O4 nanoparticles
M
Surface modification methods have been developed to modify the surface of pristine Fe3O4 nanoparticles via loading of other chemicals or biological materials during or after the process
to
improve
the
dispersibility,
stability,
biocompatibility
and
ed
synthesis
biodegradability for specific purposes [3,4]. Tannic acid, a natural polyphenolic molecule
ce pt
consisting of acentral core of glucose linking to ten gallic acid groups throughester bonds, exists in a variety of plants [5,6]. The numerous hydroxyl groups can interact with structures and biopolymers suchas some proteins, digestive enzymes, carbohydrates, and minerals to
Ac
provide many intriguing physical and chemical properties of TA-based materials [7-9].
Immobilization of enzymes on solid supports can enhance enzyme stability and proteolysis efficiency as well as it prevents the enzyme contamination in the product and, enhances the recovery for reuse [10]. Along with the extraordinary development of nanotechnology, various nanostructures were explored as carrier material for immobilization of enzymes and improvement of respective activities [11–15]. Besides, the composition of the enzyme carriers
Page 5 of 36
6 plays an important role in biocompatibility, which has a pivotal role in keeping the enzyme structure unchanged by yielding enhanced activity and efficiency [16,17].
Proteases are widely used in industrial and biomedical applications, among which trypsin is
ip t
most extensively concerned. Trypsin is a pancreatic protease, and it cleaves peptide, ester, and amide bonds at the carboxylic side of arginine, lysine and ornithine residues. Of all the
us
catalyze hydrolysis of proteins in the biochemical reactions [18].
cr
digestive endopeptidases, trypsin has the most pronounced substrate specificity, and is used to
an
Protein digestion by using trypsin is of great importance in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and
M
nonautomated due to the poor stability and autolysis of trypsin [19]. Trypsin digestion has been one of the most widely used biological reactions for both research and industrial
ed
applications [20-22]. However, the rapid autolysis of trypsin in the solution leads to unwanted and interfering fragments and, consequently decreasing the efficiency and rate of catalytic
ce pt
reactions [23,24]. Therefore, trypsin immobilization can overcome these problems.
In our previous work [25], Fe3O4 MNPs were synthesized through the co-precipitation of Fe2+
Ac
and Fe3+ in an ammonia solution. Trypsin was immobilized on the tannic acid coated Fe3O4 MNPs via non-covalent immobilization. In addition, the influences of pH, temperature, thermal stability, storage time stability and reusability were studied. The results indicated that the activity of immobilized trypsin was predominantly preserved compared to the activity of free trypsin.
Page 6 of 36
7 Herein, Fe3O4 MNPs were prepared by solvothermal method and Fe3O4 MNPs were functionalized with tannin. Since the solvothermal method enable tailoring particle size and shape to fit appropriate applications, it can be preferable in nanoparticle synthesis [4,26]. Also, covalent immobilization of trypsin on tannin modified Fe3O4 MNPs was carried out by
ip t
the formation of Schiff-base reaction or Michael-type addition between the quinone groups on tannin modified nanoparticles, which result from the pH increment, and the amino groups of
cr
the trypsin. Finally, the prepared trypsin-linked Fe3O4 MNPs were successfully utilized for the
us
digestion of BSA. The illustration of the process was given in Scheme 1. The hydrolysis products of BSA and egg white proteins formed in the presence of immobilized trypsin were
procedures.
In
addition,
the
digests
an
analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were
identified
by
matrix-assisted
laser
M
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we aimed to produce facile synthesized, non-toxically modified, and strongly trypsin
ed
immobilized magnetic nanoparticles, which exhibit a high efficiency for the protein digestion. The fragments of BSA hydrolsed by immobilized trypsin were determined and identified via
ce pt
MALDI-TOF MS.
Ac
2. Materials and methods
2.1. Materials
Ferric chloride hexahydrate (FeCl3·6H2O, >99%), sodium acetate, di-sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium dodecyl sulphate, Folin-Ciocalteu’s phenol reagent, sodium hydroxide (≥97%), ethanol (>99.2%) were procured from Merck (Germany). Trypsin from bovine pancreas, Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA, 98%), albumin from bovine serum (BSA; 98%, agarose gel electrophoresis),
Page 7 of 36
8 Bradford Reagent, benzaimidine, 4-nitroaniline (p–nitroaniline), Tetramethylethylenediamine (TEMED), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), β-merkaptoetanol (99.0%), Tris(hydroxymethyl)aminomethane, acetic acid, methanol (>99.8%) were purchased from Sigma-Aldrich (USA). TGX Stain-FreeTM FastCastTM Acrylamide Starter Kit, 10% was
Protein
Ladder
(Protein
Gel
Electrophoresis)
was
ip t
purchased from Bio-Rad Laboratories (USA). PageRuler™ Unstained Low Range obtained
from
Thermo
cr
Scientific™ (USA). Human Serum sample was obtained from Sakarya University Medico-
us
Social Center. Ethylene glycol (EG) (99%, C2H6O2) and ammonium hydrogen carbonate were provided from Tekkim (Turkey). Deionized water (DW, with resistance >18MΩ/cm) was
an
produced by using Millipore Milli Q Plus purification system, and was used throughout this
M
work.
ed
2.2. Synthesis of Fe3O4 nanoparticles and functionalization with tannin
The magnetic Fe3O4 MNPs were prepared according to a reported solvothermal method
ce pt
[27,28]. Typically, FeCl3· 6H2O (4.440 g) and NaAc (14.40 g) were first dissolved in ethylene glycol (160 mL) and the mixture was stirred at 200 rpm for 1 h. Afterward, the solution was transferred to a 50 mL Teflon-lined stainless-steel autoclave. The autoclave was sealed and
Ac
heated at 200°C and maintained for 8 h. Then, it was allowed to cool to room temperature. The magnetic Fe3O4 MNPs were washed with ethanol and deionized water several times and separated by magnetic decantation, and washed with deionized water and ethanol thrice. Finally, the product was dried in vacuum at 70 °C for 12 h.
For the preparation of tannin coated Fe3O4 MNPs (Fe3O4–T), the as-synthesized Fe3O4 MNPs (2.9708 g) were dispersed in deionized water (60 mL) by ultrasonication for 15 min and
Page 8 of 36
9 mixed at 200 rpm under N2 atmosphere at 40 °C for 1 h. Tannin (1.4862 g) was dissolved in 40 mL of deionized water and was added into the Fe3O4 nanoparticles dispersion. The mixture was stirred at 200 rpm under N2 atmosphere at 40 °C for 2 h. Then, tannin coated Fe3O4 MNPs were separated using a neodymium magnet, and washed with deionized water and
ip t
ethanol thrice, after which the product was dried in vacuum at 70 °C for 12 h [25].
us
cr
2.3. Trypsin immobilization
0.5 g of as-prepared tannin coated Fe3O4 MNPs were subjected to 3 mL of sodium hydroxide
an
(0.1 M, pH 9.4) to impart the quinone group on the tannin coated nanoparticles by using the pH dependent oxidation reaction of polyphenols [29]. The resulting solution was stirred at
M
200 rpm for 1 h at room temperature. Then, the product was separated magnetically. Subsequently, 10 mg/mL trypsin solution (containing 6 mg/mL benzamidine in 0.1 M, pH 7.5
ed
sodium phosphate buffer solution, PBS) was added into base treated modified Fe3O4 MNPs. The mixture solution was stirred at 200 rpm at 4 °C for 3 h. The product was then separated
ce pt
magnetically, and was washed with the same buffer solution for three times. The resulting enzyme immobilized Fe3O4–T (Fe3O4–T–TR) was stored at 4 ◦C until use.
Ac
2.4. Digestion of BSA and Human Serum
Standard stock BSA solution (2 mg/mL) was prepared by dissolving 20 mg BSA in 10 mL NH4HCO3 solution (100 mM, pH 8). Prior to the digestion, BSA was denatured in a 95 °C water bath for 15 min to increase efficiency of protein cleaving [30,31]. 250 µL of stock solution of BSA and 750 µL NH4HCO3 solution (100 mM, pH 8) were mixed to obtain a BSA concentration of 0.5 mg/mL.
Page 9 of 36
10 10 mg immobilized trypsin was transferred into a 1.5 mL Eppendorf tube and then 100 µL of denaturated, and diluted BSA solution was added to the same tube (100 µg/µL). Then the mixture solution was incubated at 37 °C for 1 min, 5 min and 15 min, followed by the magnetically separation using a neodymium magnet. The liquid phase was transferred to an
ip t
Eppendorf tube for MALDI-TOF MS analysis. For comparison, 50 µL solution of denaturated BSA was transferred into an Eppendorf tube, and 10 µL trypsin solution containing 6 mg/mL
cr
benzamidine in NH4HCO3 solution (0.1 M, pH 8.0) with a concentration of 5 mg/10 mL was
us
added to the same tube. After the mixture was incubated in a 37 °C for 18 hours, digestion was quenched by the addition of 11 µL of glacial acetic acid. Then the mixture was examined
M
investigated according to the same method.
an
by MALDI-TOF MS. Also, human serum (6.7 mg/dL determined by Biuret method) was
As for the microwave-assisted BSA digestion, the similar method was conducted for free and
ed
immobilized trypsin except that after microwave irradiation (CEM Mars model) with a controlled power of 700 W for 15 s, the mixture solution was separated magnetically [32] and
ce pt
the liquid phase was transferred to an Eppendorf tube for MALDI-TOF MS.
Ac
2.5. MALDI-TOF MS procedure and data analysis
MALDI mass spectra were acquired by a Voyager-DE Pro MALDI-TOF mass spectrometer (Applied Biosystems, USA). Sample desorption and ionization was carried out using the 337 nm output from a pulsed nitrogen laser (Spectra Physics, USA) at ca. 2.0×10−7 Torr vacuum and acceleration voltage of 25 kV. All spectra were acquired by averaging between 100 laser shots. 10 mg of α-cyano-4-hydroxycinnamic acid and sinapinic acid were separately dissolved in 1 mL of CH3CN:water (3:1, v/v) mixture containing 0.1% (v/v) trifluoroacetic acid.
Page 10 of 36
11 Analyte solutions were mixed with matrix in 1:10 ratio (v/v) and 1 μL of this final mixture was directly spotted onto the MALDI plate. The analysis of the digested BSA samples was acquired using sinapinic acid matrix after air-drying of each spot [33]. The mascot MS/MS ions search (Mascot database search (www.matrixscience.com) parameters were as follows:
ip t
trypsin chosen as enzyme, swissprot chosen as database, peptide mass tolerance was set to ±0.4 Da and the MS/MS tolerance was set to ±0.2 Da, seven miscleavage of trypsin enzyme
cr
specificity was allowed, peptide charge state was set to +2, +3, and +4 and carbamidomethyl
us
was set as a variable modification. Peptide matches with significant homology (p
