Journal of Proteomics & Bioinformatics

Journal of Proteomics & Bioinformatics
Open Access

ISSN: 0974-276X

+44 1223 790975

Research Article - (2008) Volume 1, Issue 7

Development of Core-shell Magnetic Mesoporous SiO2 Microspheres for the Immobilization of Trypsin for Fast Protein Digestion

Dawei Qi1, Yonghui Deng1, Yingchao Liu2, Huaqing Lin1, Chunhui Deng1*, Yan Li1, Xiangmin Zhang1, Pengyuan Yang1 and Dongyuan Zhao1
1Department of Chemistry, Fudan University, Shanghai 200433, China
2Shanghai Neurosurgical Center, Department of Neurosurgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai 200040, China
*Corresponding Author: Chunhui Deng, Department of Chemistry, Fudan University, Shanghai 200433, China, Fax: 86-21-65641740

Abstract

In the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell (designated Fe3O4@nSiO2@mSiO2) as the novel substrate for the immobilization of large amount of trypsin and applied it for fast protein digestion. Firstly, Fe3O4@nSiO2@mSiO2 microspheres were synthesized. Then, the surface of the microspheres was functionalized with GLYMO for enzyme immobilization.The amount of trypsin immobilized on Fe3O4@nSiO2@mSiO2 was about 188 ? g/mg, which was much more than that on the previous magnetic materials. Using the trypsin-immobilized magnetic mesoporous SiO2 microspheres, proteins in samples were fast digested with microwave irradiation. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analysis of the peptide fragmentation of three standard proteins, including cytochrome c (Cyt-c), myglobin (MYG), and bovine serum albumin (BSA). The functionalized magnetic microspheres served not only as substrate for enzyme immobilization, but also as excellent microwave absorbers, thus greatly improved the efficiency of protein digestion. It is also worth noting that by using this novel approach, the protein can be effectively digested within seconds, in contrast to hours required by conventional methods. Moreover, the trypsin-immobilized magnetic mesoporous SiO2 microspheres exhibit better stability than conventional methods. Furthermore, the feasibility of using this novel strategy for real sample analysis was demonstrated by applying it to the analysis of human pituitary extraction which opens a route for its further application in large-scale proteomic analysis.

Keywords: Mesoporous SiO2 microspheres ; Peptide mapping analysis ;Microwave-assisted digestion ; MALDI-TOF MS

Introduction

Proteomic analysis of complex protein mixtures usually proceed along with either bottom-up or top-down approach. In both approaches, to obtain detailed structural information, proteins are selectively cleaved into smaller polypeptide fragments by controlled chemical or enzymatic reactions (Washburn et al., 2001; Wolters et al., 2001; Zhu et al., 2003). The resulting mixture is then analyzed by MALDIMS or LC-ESI-MS. This protein analysis method is known as peptide mapping. With the progress of mass spectrometry, proteins can be rapidly identified. However, the conventional Dawei Qia, Yonghui Denga, Yingchao Liu b, Huaqing Lina, Chunhui Denga*, Yan Lia, Xiangmin Zhanga, Pengyuan Yanga, Dongyuan Zhaoa aDepartment of Chemistry, Fudan University, Shanghai 200433, China. bShanghai Neurosurgical Center, Department of Neurosurgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai 200040, China digestion method, solution based digestion, is considered to be slowly (more than hours), and presents a number of problems that may limit the speed of large scale protein identification. On the contrary, several reports have demonstrated the feasibility of protein digestion using immobilized enzyme in recent years. Immobilized enzyme is more resistant to the unfolding of their native structure that may be caused by heat and pH changes. Furthermore, they avoid contamination of the digestion products by free enzyme molecules, peptides, which can be very detrimental to the analysis (Nalivaeva et al., 2001). The immobilized enzyme has been adopted to characterize the proteins with benefits from the reusability and stability of enzyme, the higher digestion efficiency of protein analytes, and no enzyme autolysis products (Dogruel et al., 1995; Nelson, 1997; Gobom et al., 1997; Jiang et al., 2000; Ekstrõm et al., 2000; Peterson et al., 2002; Licklider et al., 1995; Ma et al., 2007; Svec, 2006). The main approach of enzyme immobilization is covalent binding. Epoxide is a classical tool for protein immobilization due to its versatile chemistry (Tischer and Wedekind, 1999; Petro et al., 1996). Petro and coworkers made the first attempt to immobilize trypsin on organic monoliths with epoxide groups in the late 1990s (K venková et al., 2005).And in many other reported approaches (K venková et al., 2005; Luo et al., 2002), the epoxide functional groups for enzyme immobilization are applied. As the authors demonstrated, the organic monoliths functionalized with epoxide can immobilize enzyme efficiently, but the procedure that modified the capillary is complicated. The most important one is that the amount of enzyme located on organic monoliths is limited, so the digestion efficiency is not satisfactory.

Recently, several approaches have been developed for fast protein digestion. One promising approach is microwaveassisted protein enzymatic digestion (MAPED). The primary advantages of MAPED are the speed and convenience. Microwave irradiation gives an acceleration of enzymatic digestion of proteins (Bose et al., 2002; Pramanik et al., 2002; Sun et al., 2006). Juan et al., (2005) used microwave technology to digest several known proteins in gel with trypsin in 5 min (Juan et al., 2005). Pramanik et al., 2002 also applied microwave technology to digest known proteins in solution or gel with trypsin in 10 min including one protein that was tightly folded and extremely resistant to denaturation (bovine ubiquitin) (Pramanik et al., 2002). More recently, Chen and Chen (2007) found that MAPED could be further accelerated by magnetite microspheres, which had been proved to be excellent absorbers of microwave radiation (Chen and Chen, 2007). Magnetic microspheres possess the unique magnetic responsivity, which means that the magnetic microspheres are not only available as highly dispersed suspensions in a wide range of sizes (50 nm to 5 mm) and permit modification on their surface, but also offer the advantage of straightforward and fast handling with the help of an applied magnetic field. With all these advantages, magnetic microspheres can be significant substrate to immobilize enzyme.

Mesoporous silica SiO2 shows great advantages as a substratum for enzyme immobilization due to its sufficient functional groups for further grafting or attachment, special surface hydrophilic/hydrophobic microregion distribution as well as stable colloidal properties. Another advantage which makes the mesoporous SiO2 more attractive for enzyme immobilization is that it possesses high specific surface area and capability of absorbing high amount of enzyme with the retention of physiological function. Recently, Zhao and Yang have successfully fabricated enzymatic reactors based on mesoporous SiO2 for proteome analysis (Fan et al., 2005; Qiao et al., 2008). In our previous work (Lin et al., 2008; Lin et al., 2008; Li et al., 2007; Li et al., 2007; Li et al., 2007; Li et al., 2007), we have successfully immobilized enzyme onto various kinds of magnetic microspheres and utilized them for facile and quick protein digestion such as on-plate digestion, in-microchip digestion and microwaveassisted digestion. However, the enzyme amount (20-70 mg/ g) on these magnetic microspheres is low. New magnetic microsphere with the ability of large amount of enzyme immobilization is desirable.

More recently, a composite microsphere consisting of Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell (designated Fe3O4@nSiO2@mSiO2) with large surface areas was successfully synthesized and applied in environmental analysis (Deng et al., 2008). Herein, we report the feasibility of combining the advantages of mesoporous SiO2, magnetic microspheres and microwaveassisted protein enzymatic digestion to develop a facile and highly efficient proteolysis strategy. Glycidoxypropyltrimethoxysilane (GLYMO) - functionalized Fe3O4@nSiO2@mSiO2 was successfully synthesized for trypsin immobilization and fast microwave assisted digestion. High digest efficiency can be achieved in 20 sec for both standard proteins and human pituitary extract. This approach greatly shortens and simplifies the digestion process and provides a promising way to facilitate the complete automation of top-down proteomic approaches for large-scale analysis.

Materials and Methods

TFA was purchased from Merck (Darmstadt, Germany). ACN was HPLC grade from Fisher Scientific (Fairlawn, NJ, USA). Bovine serum albumin (BSA, fraction V) was obtained from Bio Basic Inc (Toronto, Canada). (TPCK)- treated trypsins, cytochrome c (EC 232-700-9), myglobin were purchased from Sigma Chemical (St. Louis, MO). Sinapinic acid and cyano-4-hydroxycinnamic 6 acid (CHCA) were purchased from Sigma (St. Louis, MO, USA). Water was purified using a Milli Q system (Millipore, Molsheim, France). All of the other chemicals were of analytical grade and used as received.

Synthesis of GLYMO-functionalized Fe3O4@ nSiO2 @mSiO2 magnetic mesoporous SiO2 microspheres

Synthesis of Fe3O4@nSiO2 magnetic microspheres The magnetic microspheres were synthesized through solvothermal reaction as described in our previous work with some modification (Xu et al., 2006). Briefly, 2.70 g of FeCl3·6H2O was first dissolved in 100 mL of ethylene glycol under magnetic stirring. A yellow clear solution was obtained after stirring for 0.5 h. Then 7.20 g of NaAc (sodium acetate) was added to this solution. After being stirred for another 0.5 h, the resultant solution was transferred into a Teflon-lined stainless-steel autoclave with capacity of 200 mL. The autoclave was sealed and heated at 200°C for 16 h and cooled to room temperature. The black magnetic microspheres were collected with the help of a magnet filed, followed by washing with recycle of ethanol and deioned water for six times. The product was then dried in vacuum at 60°C for 12 h.

In order to obtain core-shell magnetic silica microspheres with narrow size distribution and uniform thickness of silica via sol-gel approach, magnetic microspheres (0.01 g) were first treated in HCl aqueous solution (5.0 mL, 2 M) under ultrasonic vibration for 5 min. Then, the microspheres were thoroughly washed with deioned water and redispersed in a mixture of ethanol (70.0 g), deioned water (20.0 g), and concentrated ammonia aqueous solution (1.0 g, 28 wt %) with the help of ultrasonication, and a stable dispersion was obtained. Subsequently, tetraethyl orthosilicate (TEOS) (0.05 g) was added to the above dispersion under mechanistic stirring, and the reaction was allowed to proceed for 12 h. Finally, by the use of a magnet, the product was separated, washed with ethanol and water, and then vacuum dried at 60°C for 24 h.

Synthesis of Fe3O4@nSiO2@mSiO2 magnetic mesoporous SiO2 microspheres

According to our previous method (Deng et al., 2008), the synthesis of Fe3O4@nSiO2@mSiO2 magnetic mesoporous SiO2 microspheres were performed. At first, the Fe3O4@nSiO2 microspheres were redispersed in a mixed solution containing acetyl trimethyl ammonium bromide (CTAB) (0.30 g, 0.823 mmol) deioned water (80 mL), concentrated ammonia aqueous solution (1.00 g, 28 wt %) and ethanol (60 mL). The mixed solution was homogenized for 0.5 h to form a uniform dispersion. 0.40 g of TEOS (1.90 mmol) was added dropwise to the dispersion with continuous stirring. After the reaction for 6 h, the product was collected with a magnet and washed repeatedly with ethanol and water to remove nonmagnetic by-products. Finally, the purified microspheres were redispersed in 60 mL of acetone and refluxed at 80°C for 48 h to remove the template CTAB. The extraction was repeated for three times, and the microspheres were washed with deioned water, and Fe3O4@nSiO2@mSiO2 microspheres were finally produced.

Synthesis of GLYMO-functionalized Fe3O4@nSiO2@mSiO2 magnetic mesoporous SiO2 microspheres

80 mg of Fe3O4@nSiO2@mSiO2 microspheres were redispersed in 20 ml methylbenzene containing 0.35 ml GLYMO with the help of ultrasonication. Subsequently, the suspension was refluxed at 80°C for 12 h. Finally, the microspheres were washed with ethanol three times, and then vacuum dried at 60°C for 24 h.

Immobilization of trypsin to the GLYMO-functionalized Fe3O4@nSiO2@mSiO2

For enzyme immobilization (Scheme 1), 1.0 mg of GLYMO-functionalized Fe3O4@nSiO2@mSiO2 microspheres was incubated with TPCK-treated trypsin (0.1 mL; 5 mg/mL) for 1 h under gentle rotation. After removal of the excess trypsin solution, the trypsin immobilized GLYMO-functionalized Fe3O4@nSiO2@mSiO2 microspheres were washed with 25 mM NH4HCO3 (4 × 200 μ L). The final product was stored in 25 mM NH4HCO3 at 4oC before use.

After the trypsin immobilization, the microspheres were retained by a magnet, and the UV absorption value of the supernatant solution was measured at λ= 280 nm to calculate the amount of trypsin immobilized on the GLYMOfunctionalized Fe3O4@nSiO2@mSiO2 microspheres.

Microwave-assisted protein digestion

The procedure of tryptic digestion using trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres is shown in Scheme S2. Three standard proteins, cytochrome c (Cyt-c), myglobin (MYG) and bovine serum albumin (BSA), in 25 mM NH4HCO3 buffer solution (pH 7.7), were used as model substrate to evaluate the digestion performance. The trypsin-immobilized microspheres were transferred into 40 μL protein solution (0.20 μg/μL) in a 0.6mL Eppendorf tube. A domestic microwave oven (output power 700 W) was used to conduct the microwave-assisted protein digestion process. After microwave irradiation, using an external magnet to retain the magnetic microspheres, the supernatant was deposited onto a MALDI plate directly.

In-solution enzymatic digestion

For comparison, the digestions of Cyt-C, MYG and BSA were also performed by free trypsin in solution according to the conventional procedure. The standard proteins were firstly denatured in 25 mM NH4HCO3 buffer containing 8 M urea for 1 h at 37°C, followed by dilution with 25 mM NH4HCO3 (pH 7.7) buffer to the concentration of urea below 1 M. The in-solution digestion was performed by adding trypsin into the protein solution at a substrate-toenzyme ratio of 40:1, and the solution was incubated at 37°C for 12 h. After digestion, 1.0 μ L of formic acid was added into the solution to stop the reaction.

Extraction of human pituitary

According to the references (Li et al., 2007; Che et al., 2005; Zhan et al., 2006; Liu et al., 2006), the extraction of proteins in human pituitary was performed as the following protocol. The human pituitary tissue was cleaned with Milli-Q water to remove some possible contaminants, cut into small pieces, and homogenized in water containing 9.0 M urea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), and 1.0 mM phenylmethylsulfonyl fluoride (PMSF) using a glass vessel in an ice bath. The resulting homogenate was swirled for 30 min and centrifuged for 20 min at 18000 g. The supernatant was collected, fractionated in aliquots, and stored at -80°C till further analysis. Protein concentration was measured using the Bradford assay using BSA as standard, 20 μg/μL for human pituitary tissue.

Mass Spectrometry and Database Searching MALDITOF MS Analysis

Sample solutions were deposited on the MALDI target using the dried droplet method. An amount of 1 μL of sample solution was spotted onto the MALDI plate, and then another 0.5 μL of CHCA matrix solution (5 mg/mL, 0.1% TFA in 50% ACN/H2O solution) was introduced. Positive ion MALDI-TOF-MS spectra were acquired on a 4700 Proteomics Analyzer (Applied Biosystems). The sample was excited using an Nd:YAG laser (355 nm) operated at a repetition rate of 200 Hz and acceleration voltage of 20 kV. Before identifying the samples, the MS instrument was calibrated by an internal calibration with tryptic peptides of myoglobin. The MASCOT server was used to interpret the MALDI-TOF MS data by searching the species of Mammals from sprot-horse for identification of three standard proteins with peptide fingerprint mass spectra.

LC-ESI-MS/MS Process

The elution gradient for the RPLC column was from 5 to 90% buffer B (0.1% formic acid, 95% ACN). Eluted peptides were detected in a survey scan from 400 to 1800 amu (1 microscan) followed by 8 data-dependant MS/MS scans in a completely automated fashion on an LTQ-Orbitrap ESI mass spectrometer. According to Washburn's method (Washburn et al., 2001), the filtering criteria was calculated through a reverse database searching and the Xcorr value vs charges was obtained as following: p< 0.01, >2.78(+3),>2.10(+2), >2.0(+1); ≥ Cn> 0.1 and peptide length > 7 were also applied.

Result and Discussion

Preparation of GLYMO-functionalized Fe3O4 @nSiO2 @mSiO2 microspheres

The Fe3O4@nSiO2@mSiO2 microspheres were synthesized according to previously reported approach (Figure 1) which involves first coating Fe3O4 paritcles (~ 300 nm in diameter) with nonporous silica layer and then with mesoporous silica layer by using organic surfactant as the templates. Transmission electron microscopy (TEM) image shows that the obtained Fe3O4@nSiO2@mSiO2 microspheres have a mean diameter of about 500 nm and possess well-defined silica-coated magnetite core and mesoporous silica shell (Figure 1a). The nonporous silica layer is 20 nm in thickness, which can serve as protective coating for magnetite, and the mesoporous silica layer is 70 nm in thickness, which can provide the microspheres with high surface area for derivation of numerous functional groups. Notably, the mesopores (~ 2.0 nm in diameter) in the shell were found to be cylindrical and perpendicular to the microsphere surface, which provide good accessibility for reactants. Scanning electron microscophy (SEM) images show that the microspheres are very uniform both in size and shape (Figure 1b). The unique microstructure of the obtained microspheres would be very useful for many applications. According to our method (Lin et al., 2008), GLYMO-functionalized Fe3O4@nSiO2@mSiO2 magnetic mesoporous silica microspheres were prepared by modifying their surface with a silane coupling agent GLYMO.

proteomics-bioinformatics-autocatalytic

Figure 1: Illustration of synthesis of trypsin-immobilized Fe O @nSiO @mSiO magnetic microspheres.

proteomics-bioinformatics-immobilized

Figure 2: MALDI-TOF MS spectra of tryptic peptides originated from cytochrome c, myoglobin, and bovine serum albumin resulted from microwave-assisted digestion by trypsin-immobilized Fe O @nSiO @mSiO magnetic microspheres.*, peptide from standard proteins.

Immobilization of trypsin

The most important factor of conventional enzymatic proteolysis is the enzyme-substrate ratio, and the digestion efficiency increases observably with high enzyme-substrate ratio. Nevertheless, if a high concentration of free enzyme is used, the digestion proceeds quicker but autolysis products become more abundant, and enzyme autoproteolysis would impair signal interpretation, especially for low amounts of analytes (Bonneil et al., 2000). However, the immobilization can improve the enzyme stability, and retain its activity. The mesoporous SiO2 with high specific surface can provide sufficient functional groups such as hydroxyl group for further modification, so more enzyme can be immobilized onto the channels of the microspheres. The elevated enzymesubstrate ratio on the surface leads to enhanced digestion efficiency. In the work, trypsin can be immobilized onto the functionalized magnetic microspheres only through a onestep reaction of its amine group with GLYMO group. As the procedures that mentioned above the amount of trypsin immobilized on the magnetic microspheres was about 188 μg/mg, which was much more than that on the previous magnetic materials including commercial magnetic materials (Lin et al., 2008; Lin et al., 2008 ; Li et al., 2007; Li et al., 2007; Li et al., 2007; Li et al., 2007). The protein to enzyme ratio is about 1:5 as compared to 40:1 in in-solution digestion procedure.

Microwave-assisted protein enzymatic digestion

The procedure of tryptic digestion using trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres is shown in Scheme S2. As we know, in many references [Juan et al., 2005; Chen and Chen, 2007; Fan et al., 2005; Qiao et al., 2008], MYG (MW 16 700) or Cyt-C (MW 12 384) and BSA (MW 66 000) was used as model proteins to test the digestion efficiency. In our work, the three proteins were also used. For comparison, the digestions were also performed by free trypsin in solution for 12 h. Cyt-C and MYG is small molecule proteins which would be proteolysis easily, and were used to investigate the feasibility of our new method. The peptide mass mapping of Cyt-C and MYG from microwave-assisted digestion were displayed in Figure 1. Many digest fragments were observed from the MS spectra suggesting a highly proteolytic efficiency for the Fe3O4@nSiO2@mSiO2 microspheres. Notably, there are no distinct peaks with m/z > 2500, indicating virtually complete digestion. The proteolytic results were listed in Table 1 in detail. The observation corresponded to the detection of fragments containing 81 out of 104 possible amino acids of Cyt-C, 137 out of 153 possible amino acids of MYG. The sequence coverage obtained from the database is 77% for Cyt-C, 89% for MYG. Therefore, we can confirm that our new approach is feasible for fast protein digestion. Then BSA with larger molecule weight was used to do further investigation of our method. The observation corresponded to the detection of fragments containing 263 out of 607 possible amino acids of BSA, and the sequence coverage is 45% for BSA. The results indicate that BSA has been digested completely, which means that the new method is suitable for large protein molecule digestion. The identification results are comparative with those by in solution digestion that required a reaction time of 12 h (Table 1). Meanwhile, the sample volume is only 40 μ L per analysis. Moreover, no trypsin autolysis peaks were observed from mass spectra in the microwave-assisted digestion method, which demonstrated that enzyme immobilization technique can overcome the trypsin autolysis (Dogruel et al., 1995; Nelson, 1997; Gobom et al., 1997; Jiang et al., 2000; Ekstrõm et al., 2000; Peterson et al., 2002; Licklider et al., 1995; Ma et al., 2007; Svec, 2006). Since the magnetic microspheres are excellent microwave absorbers; and thus greatly improve the efficiency of protein digestion.

protein Cyt-c BSA MYG
  microwave in-
solution
microwave In-
solution
microwave in-
solution
amino acids identified 81 79 263 213 137 107
sequence
coverage (%)
77 75 45 35 89 69
peptides
matched
12 10 27 18 13 10
accession no. P00004 P00004 P02769 P02769 P68083 P68083
protein MW 11694.1 11694.1 69248.4 69248.4 16941 16941

Table 1: MALDI-TOF/TOF MS data of digestion products by microwave-assisted digestion using trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheresa
a, Three spot replicates were taken in the experiments.

Cyt-c MYG BSA
9-22
IFVQKCAQCHTVEK
26-38
HKTGPNLHGLFGR
28-38
TGPNLHGLFGR
28-39
TGPNLHGLFGRK
39-53
KTGQAPGFTYTDANK
40-53
TGQAPGFTYTDANK
40-55
TGQAPGFTYTDANKNK
56-72
GITWKEETLMEYLENPK
61-72
EETLMEYLENPK
61-73
EETLMEYLENPKK
80-87
MIFAGIKK
89-99
TEREDLIAYLK
1-31
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIR
17-31
VEADIAGHGQEVLIR
32-42
LGTGHPETLEK
32-45
LGTGHPETLEKFDK
48-56
HLKTEAEMK
64-77
HGTVVLTALGGILK
79-96
KGHHEAELKPLAQSHATK
80-96
GHHEAELKPLAQSHATK
103-118
YLEFISDAIIHVLHSK
103-133 YLEFISDAIIHVLHSKHPGDFGADAQGA MTK 119-133 HPGDFGADAQGAMTK 134-145 ALELFRNDIAAK 146-153
YKELGFQG
25-34
DTHKSEIAHR
35-44
FKDLGEEHFK
65-75
LVNELTEFAK
89-105
SLHTLFGDELCKVASLR
157-167
FWGKYLYEIAR
161-167
YLYEIAR
161-168
YLYEIARR
168-183 RHPYFYAPELLYYANK
198-209
GACLLPKIETMR
221-228
LRCASIQK
341-359
NYQEAKDAFLGSFLYEYSR
347-359
DAFLGSFLYEYSR
347-360
DAFLGSFLYEYSRR
360-371
RHPEYAVSVLLR
361-371
HPEYAVSVLLR
402-412
HLVDEPQNLIK
421-433
LGEYGFQNALIVR
437-451 KVPQVSTPTLVEVSR
438-451
VPQVSTPTLVEVSR
456-468
VGTRCCTKPESER
469-482
MPCTEDYLSLILNR
508-523
RPCFSALTPDETYVPK
529-544
LFTFHADICTLPDTEK
569-580
TVMENFVAFVDK
588-597
EACFAVEGPK

Table 2: Detail-identified fragments of Cyt-C, MYG and BSA by MALDI-TOF/TOF MS

We compared the digestion efficiency of our protocol with other reported methods (Chen and Chen, 2007; Fan et al., 2005; Zhang et al., 2006; Guo et al., 2003), and the results were listed in Table S2 (see supporting information). The digestion time speared from 20 s to 15 min with different digestion method, and the sequence coverage ranges from 77% to 89% of cytochrome c and 44% to 89% of myglobin. This demonstrates that our method shows comparable digestion efficiency with other methods, and our method takes short digestion time (only 20s).

The effect of different addition amounts of trypsinimmobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres on the sequence coverage of MYG is shown in Figure 3.

proteomics-bioinformatics-microspheres

Figure 3: Effect of microspheres amount on sequence coverage of MYG digests resulted from microwave-assisted digestion by trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres.

The sequence coverage of MYG increases slowly when the addition amounts are less than 200 μg. Increasing the amounts of microspheres does not change the sequence coverage distinctly. It suggests that the optimal addition amounts of the microspheres are 200 μg. Figure 4 shows the influence of incubation time on sequence coverage of MYG obtained from MALDI-TOF MS analysis. When the incubation time increases from 5 to 20 sec, the sequence coverage accordingly increases from 78 to 89%. With further increasement of the time, no significant change on sequence coverage is observed, suggesting that 20 s is enough for efficient microwave-assisted digestion.

proteomics-bioinformatics-incubation

Figure 4: Effect of incubation time on sequence coverage of MYG digests resulted from microwave-assisted digestion by trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres.

The digestion time by the microwave-assisted digestion was much less than that (5 min) in trypsin-immobilized magnetic microspheres without microwave assistance (Li et al., 2007). Figure 5 distinctly. Therefore, we concluded that 200 μg microspheres with microwave incubation at 700 W for 20 sec is the optimal condition for effective protein digestion. This shows that microwave-assisted digestion by using trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres has very high digestion efficiency.

proteomics-bioinformatics-microspheres

Figure 5: Effect of microwave power on sequence coverage of MYG digests resulted from microwave-assisted digestion by trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres.

The high digestion efficiency of the procedure may mostly count on two reasons: firstly, the mesoporous SiO2 with high specific surface can provide sufficient functional groups such as hydroxyl group for further modification, so more enzyme can be immobilized onto the channels of the microspheres. The elevated enzyme-substrate ratio on the surface leads to enhanced digestion efficiency. Secondly, the functionalized magnetic microspheres served not only as substrate for enzyme immobilization, but also as excellent microwave absorbers, thus greatly improved the efficiency of protein digestion.

To investigate dynamic range of protein by the proposed microwave-assisted digestion, 200 mg of trypsin-immobilized magnetic microspheres were added into 40 μl of MYG solution with the concentration of 100 ng/ μl, 50 ng/ μl, 20 ng/ μl, 10 ng/ μl respectively. The microwave-assisted digestion was conducted in the same conditions described as above. The protein sequence coverage of the four protein concentration (10 to 100 ng/μl) is 73%, 79%, 79%, 83%, respectively. The results indicated that the proposed approach can be used for fast digestion of low concentration of proteins

To test the stability, seven consecutive operations for MYG with incubation for 20 sec using the trypsin-immobilized microspheres were conducted. As shown in Figure 6, no obvious decrease is observed in the runs, suggesting that the activity of the enzyme immobilized is not destroyed apparently.

proteomics-bioinformatics-microwave

Figure 6: Stability test of microwave-assisted digestion by trypsin-immobilized Fe3O4@nSiO2@mSiO2 magnetic microspheres

Furthermore, we studied the longevity of the enzyme-immobilized microspheres, we used 200 mg of the enzyme-immobilized magnetic microspheres for microwaveassisted digestion in the same conditions described as above. Then, the microspheres were washed with 25 mM NH4HCO3 (4×200μl), and resuspended in 200μl 25mM NH4HCO3 for further use. The same digestion procedure was conducted every other 24 h with the same microreactor. After the experiment ran four times, the protein sequence coverage didn't change obviously. It means that the activity of the protein microreactor didn't minish after 96 h. This shows that the microreactor has the longevity of more than 4 days.

Application of microwave-assisted protein enzymatic digestion

In recent decades, there has been an explosion of interest in the identification and characterization of proteins, using the techniques of mass spectrometry and database searching, with the aim of establishing links to pathological conditions. A formal step in identification of proteins is protein digestion prior to mass spectrometry analysis. Our approach provides a facile and low-cost way to produce protein fragmentations, which generate sequence information and ultimately identification.

Here, to further confirm the feasibility of microreactor for the analysis of complex protein mixtures, it was applied to human pituitary extract. Without any preparation and prefractionation procedure, the entire proteome was digested for only 1 min and went through LC-ESI-MS/MS directly. Figure 7A was the total ion chromatogram acquired from the microwave-assisted protein digestion. After a database search according to the SEQUEST criteria set above (Experimental Section), 951 peptides were identified, 589 proteins were identified with p < 0.01 (Table 1). Figure 7B is the precursor mass scan at 13.22 min and Figure 7C is the corresponding MS/MS spectrum of the m/z 1095.46 in Figure 7C. Most y-ions together with b-ions produced from the precursor ion matched together and resulted in the high reliability for peptide sequence of R.NMGGPYGGGNYGPGGSGGSGGYGGR.S. These results clearly show that this novel digestion approach can be used for large scale proteomic analysis.

proteomics-bioinformatics-chromatogram

Figure 7: (A) The total ion chromatogram for the separation of human pituitary extract digests by microwave-assisted digestion. (B)The mass spectrum scan at 13.22min in LC-ESI-MS for human pituitary digests. The ms/ms spectrum of 1095.46 m/z peak in figure 7B.

Conclusions

In this study, we successfully developed GLYMO-modified Fe3O4@nSiO2@mSiO2 magnetic mesoporous silica microspheres as a new substrate for immobilization of large amount of trypsin, and applied it to microwave-assisted protein digestion. Compared with conventional in solution digestion, microwave-assisted protein digestion based on Fe3O4@nSiO2@mSiO2 can show similar identification results with much shorter incubation time. The excellent efficiency of trypsin-immobilized microspheres can be also verified when it is applied to real proteome, human pituitary extract. At the same time, the process of digestion is very facile due to the easy manipulation of magnetic microspheres and microwave processing. Considering the combination with diversely high automated separation techniques, the novel microwave-assisted protein digestion method with trypsinimmobilized magnetic mesoporous silica microspheres developed here would hasten high-throughput proteome analysis.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (Project: 20875017 and 20871030), the National Basic Research Priorities Program (Project: 2007CB914100/3), the National High Technology Research and Development Program of China 863 Project (No. 2006AA02Z4C5), and Shanghai Leading Academic Discipline Project (B109).

References

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  2. Bose AK, Ing YH, Lavlinskaia N, Sareen C, Pramanik BN, et al. (2002) Microwave Enhanced Akabori Reaction for Peptide Analysis. J Am Soc Mass Spectrom 13: 839-850. » CrossRef   » PubMed   »  Google Scholar
  3. Che F, Lim J, Pan H, Biswas R, Fricker L D (2005) Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Mol Cell Proteomics 4: 1391- 1405.» CrossRef   » PubMed   »  Google Scholar
  4. Chen WY, Chen YC (2007) Acceleration of microwaveassisted enzymatic digestion reactions by magnetite beads. Anal Chem 79: 2394-2401. » CrossRef   » PubMed   »  Google Scholar
  5. Deng YH, Qi DW, Deng CH, Zhang XM, Zhao DY (2008) Superparamagnetic high-magnetization microspheres with Fe3O4@SiO2 coe and prpendicularly aigned msoporous SiO2 shell for removal of microcystins. J Am Chem Soc 130: 28-29. » CrossRef   » PubMed   »
  6. Dogruel D, Williams P, Nelson RW (1995) Rapid tryptic mapping using enzymically active mass spectrometer probe tips. Anal Chem 67: 4343–4348. » CrossRef   » PubMed   »  Google Scholar
  7. Ekstrõm S, Õnerfjord P, Nilsson J, Bengtsson M, Laurell T, et al. (2000) Integrated microanalytical technology enabling rapid and automated protein identification. Anal Chem 72: 286–293. » CrossRef   » PubMed   »  Google Scholar
  8. Fan J, Shui WQ, Yang PY, Wang XY, Xu YM, et al. (2005) Mesoporous silica nanoreactors for highly efficient proteolysis. Chem Eur J 11: 5391 – 5396. » CrossRef   » PubMed   »  Google Scholar
  9. Gobom J, Nordhoff E, Ekman R, Roepstorff P (1997) Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. J Mass Spectrom 32: 593-601. » CrossRef  »  Google Scholar
  10. Guo Z, Xu SY, Lei ZD, Zou HF, Guo BC (2003) Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 24: 3633-3639. » CrossRef   » PubMed
  11. Jiang HH, Zou HF, Wang HL, Zhang Q, Ni JY, et al. (2000) Combination of MALDI-TOF mass spectrometry with immobilized enzyme microreactor for peptide mapping. Science in China (B) 43: 625–633. » CrossRef  »  Google Scholar
  12. Juan HF, Chang SC, Huang HC, Chen ST (2005) A new application of microwave technology to proteomics. Proteomics 5: 840-842. » CrossRef   » PubMed   »  Google Scholar
  13. Kvenková J, Bilková Z, Fore F (2005) Chararacterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS. J Sep Sci 28: 1675 - 1684. » CrossRef   » PubMed   »  Google Scholar
  14. Li Y, Xu XQ, Yan B, Deng CH, Yu WJ, et al. (2007) Microchip reactor packed with metal-ion Chelated magnetic silica microspheres for highly efficient proteolysis. J. Proteome Res 6: 2367-2375. » CrossRef   » PubMed   »  Google Scholar
  15. Li Y, Xu XQ, Deng CH, Yang PY, Zhang XM (2007) Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis. J Proteome Res 6: 3849-3855. » CrossRef   » PubMed   »  Google Scholar
  16. Li Y, Yan B, Deng CH, Yu WJ, Xu XQ, et al. (2007) Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres. Proteomics 7: 2330-2339.» CrossRef   » PubMed   »  Google Scholar
  17. Li Y, Yan B, Xu XQ, Deng CH, Yang PY, et al. (2007) On-column tryptic mapping of proteins using metal-ionchelated magnetic silica microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 21: 2263-2268. » CrossRef   » PubMed   »  Google Scholar
  18. Licklider L, Kuhr WG, Lacey MP, Keough T, Purdon MP, et al. (1995) Online microreactors/capillary electrophoresis/ mass spectrometry for the analysis of proteins and peptides. Anal Chem 67: 4170-4177. » CrossRef  »  Google Scholar
  19. Lin S, Lin ZX, Yao GP, Deng CH, Yang PY, et al. (2007) Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Rapid Commun Mass Spectrom 21: 3910- 3918. » CrossRef   » PubMed   »  Google Scholar
  20. Lin S, Yao GP, Qi DW, Li Y, Deng CH, et al. (2008) Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres. Anal Chem 80: 3655-3665. » CrossRef   » PubMed   »  Google Scholar
  21. Lin S, Yun D, Qi DW, Deng CH, Li Y, et al. (2008) Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis. J Proteome Res 7: 1297-1307. » CrossRef   » PubMed   »  Google Scholar
  22. Liu F, Baggerman G, D'Hertog W (2006) In silico identification of new secretory peptide genes in Drosophila melanogaster. Mol Cell Proteomics 5: 510-522. » CrossRef   » PubMed   »  Google Scholar
  23. Luo QZ, Mao XQ, Huang Xm, Zou HF (2002) Highperformance affinity chromatography for characterization of human immunoglobulin G digestion with papain. J Chromatogr B 776: 139 - 147. » CrossRef   » PubMed   »  Google Scholar
  24. Ma JF, Zhang LH, Liang Z, Zhang WB, Zhang YK (2007) Monolith-based immobilized enzyme reactors: Recent developments and applications for proteome analysis. J Sep Sci 30: 3050-3059. » CrossRef   » PubMed   »  Google Scholar
  25. Nalivaeva NN, Turner A (2001) Post-translational modifications of proteins: Acetylcholinesterase as a model system. Proteomics 1: 735-747. » CrossRef   » PubMed   »  Google Scholar
  26. Nelson RW (1997) The use of bioreactive probes in protein characterization. Mass Spectrom Rev 16: 353- 376. » CrossRef   » PubMed   »  Google Scholar
  27. Peterson DS, Rohr T, Svec F, Fréchet JMJ (2002) Enzymatic microreactor-on-a-Chip: Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem 74: 4081- 4088. » CrossRef   » PubMed   »  Google Scholar
  28. Pramanik NB, Mirza UA, Ning YH, Liu YH, Bartner PL, et al. (2002) Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes. Protein Sci 11: 2677-2687. » CrossRef   » PubMed   »  Google Scholar
  29. Qiao L, Liu Y, Hudson SP, Yang PY, Liu BH (2008) A nanoporous reactor for efficient proteolysis. Chem Eur J 14: 151 - 157. » CrossRef   » PubMed   »  Google Scholar
  30. Sun W, Gao SJ, Wang LJ, Chen Y, Wu SZ, et al. (2006) Microwave-assisted protein preparation and enzymatic digestion in proteomics. Mol Cell Proteomics 5: 769-776. » CrossRef   » PubMed   »  Google Scholar
  31. Svec F (2006) Less common applications of monoliths: Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports. Electrophoresis 27: 947-961. » CrossRef   » PubMed   »  Google Scholar
  32. Tischer W, Wedekind F (1999) Immobilized Enzymes: Methods and Applications. Topic Curr Chem 200: 95 - 126. » CrossRef  »  Google Scholar
  33. Xu XQ, Deng CH, Gao MX, Yu WJ, Yang PY, et al. (2006) Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Adv Mater 18: 3289-3293. » CrossRef  »  Google Scholar
  34. Washburn MP, Wolters DA, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19: 242- 247. » CrossRef   » PubMed   »  Google Scholar
  35. Wolters DA, Washburn MP, Yates JR (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73: 5683-5690. » CrossRef   » PubMed   »  Google Scholar
  36. Zhang YH, Liu Y, Kong JL, Yang PY, Tang Y, et al. (2006) Efficient proteolysis system: A nanozeolite-derived microreactor. Small 2: 1170 - 1173. » CrossRef   » PubMed   »  Google Scholar
  37. Zhu H, Bilgin M, Snyder M (2003) Proteomics. Annu Rev Biochem 72: 783-812. » CrossRef   » PubMed   »  Google Scholar
  38. Zhan XQ, Desiderio DM (2006) Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry. Anal Biochem 354 : 279-289. » CrossRef   » PubMed   »  Google Scholar

References

  1. Bonneil E, Mercier M, Waldron KC (2000) Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis. Anal Chim Acta 404: 29–45. » CrossRef  »  Google Scholar
  2. Bose AK, Ing YH, Lavlinskaia N, Sareen C, Pramanik BN, et al. (2002) Microwave Enhanced Akabori Reaction for Peptide Analysis. J Am Soc Mass Spectrom 13: 839-850. » CrossRef   » PubMed   »  Google Scholar
  3. Che F, Lim J, Pan H, Biswas R, Fricker L D (2005) Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Mol Cell Proteomics 4: 1391- 1405.» CrossRef   » PubMed   »  Google Scholar
  4. Chen WY, Chen YC (2007) Acceleration of microwaveassisted enzymatic digestion reactions by magnetite beads. Anal Chem 79: 2394-2401. » CrossRef   » PubMed   »  Google Scholar
  5. Deng YH, Qi DW, Deng CH, Zhang XM, Zhao DY (2008) Superparamagnetic high-magnetization microspheres with Fe3O4@SiO2 coe and prpendicularly aigned msoporous SiO2 shell for removal of microcystins. J Am Chem Soc 130: 28-29. » CrossRef   » PubMed   »
  6. Dogruel D, Williams P, Nelson RW (1995) Rapid tryptic mapping using enzymically active mass spectrometer probe tips. Anal Chem 67: 4343–4348. » CrossRef   » PubMed   »  Google Scholar
  7. Ekstrõm S, Õnerfjord P, Nilsson J, Bengtsson M, Laurell T, et al. (2000) Integrated microanalytical technology enabling rapid and automated protein identification. Anal Chem 72: 286–293. » CrossRef   » PubMed   »  Google Scholar
  8. Fan J, Shui WQ, Yang PY, Wang XY, Xu YM, et al. (2005) Mesoporous silica nanoreactors for highly efficient proteolysis. Chem Eur J 11: 5391 – 5396. » CrossRef   » PubMed   »  Google Scholar
  9. Gobom J, Nordhoff E, Ekman R, Roepstorff P (1997) Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. J Mass Spectrom 32: 593-601. » CrossRef  »  Google Scholar
  10. Guo Z, Xu SY, Lei ZD, Zou HF, Guo BC (2003) Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 24: 3633-3639. » CrossRef   » PubMed
  11. Jiang HH, Zou HF, Wang HL, Zhang Q, Ni JY, et al. (2000) Combination of MALDI-TOF mass spectrometry with immobilized enzyme microreactor for peptide mapping. Science in China (B) 43: 625–633. » CrossRef  »  Google Scholar
  12. Juan HF, Chang SC, Huang HC, Chen ST (2005) A new application of microwave technology to proteomics. Proteomics 5: 840-842. » CrossRef   » PubMed   »  Google Scholar
  13. Kvenková J, Bilková Z, Fore F (2005) Chararacterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS. J Sep Sci 28: 1675 - 1684. » CrossRef   » PubMed   »  Google Scholar
  14. Li Y, Xu XQ, Yan B, Deng CH, Yu WJ, et al. (2007) Microchip reactor packed with metal-ion Chelated magnetic silica microspheres for highly efficient proteolysis. J. Proteome Res 6: 2367-2375. » CrossRef   » PubMed   »  Google Scholar
  15. Li Y, Xu XQ, Deng CH, Yang PY, Zhang XM (2007) Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis. J Proteome Res 6: 3849-3855. » CrossRef   » PubMed   »  Google Scholar
  16. Li Y, Yan B, Deng CH, Yu WJ, Xu XQ, et al. (2007) Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres. Proteomics 7: 2330-2339.» CrossRef   » PubMed   »  Google Scholar
  17. Li Y, Yan B, Xu XQ, Deng CH, Yang PY, et al. (2007) On-column tryptic mapping of proteins using metal-ionchelated magnetic silica microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 21: 2263-2268. » CrossRef   » PubMed   »  Google Scholar
  18. Licklider L, Kuhr WG, Lacey MP, Keough T, Purdon MP, et al. (1995) Online microreactors/capillary electrophoresis/ mass spectrometry for the analysis of proteins and peptides. Anal Chem 67: 4170-4177. » CrossRef  »  Google Scholar
  19. Lin S, Lin ZX, Yao GP, Deng CH, Yang PY, et al. (2007) Development of microwave-assisted protein digestion based on trypsin-immobilized magnetic microspheres for highly efficient proteolysis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Rapid Commun Mass Spectrom 21: 3910- 3918. » CrossRef   » PubMed   »  Google Scholar
  20. Lin S, Yao GP, Qi DW, Li Y, Deng CH, et al. (2008) Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres. Anal Chem 80: 3655-3665. » CrossRef   » PubMed   »  Google Scholar
  21. Lin S, Yun D, Qi DW, Deng CH, Li Y, et al. (2008) Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis. J Proteome Res 7: 1297-1307. » CrossRef   » PubMed   »  Google Scholar
  22. Liu F, Baggerman G, D'Hertog W (2006) In silico identification of new secretory peptide genes in Drosophila melanogaster. Mol Cell Proteomics 5: 510-522. » CrossRef   » PubMed   »  Google Scholar
  23. Luo QZ, Mao XQ, Huang Xm, Zou HF (2002) Highperformance affinity chromatography for characterization of human immunoglobulin G digestion with papain. J Chromatogr B 776: 139 - 147. » CrossRef   » PubMed   »  Google Scholar
  24. Ma JF, Zhang LH, Liang Z, Zhang WB, Zhang YK (2007) Monolith-based immobilized enzyme reactors: Recent developments and applications for proteome analysis. J Sep Sci 30: 3050-3059. » CrossRef   » PubMed   »  Google Scholar
  25. Nalivaeva NN, Turner A (2001) Post-translational modifications of proteins: Acetylcholinesterase as a model system. Proteomics 1: 735-747. » CrossRef   » PubMed   »  Google Scholar
  26. Nelson RW (1997) The use of bioreactive probes in protein characterization. Mass Spectrom Rev 16: 353- 376. » CrossRef   » PubMed   »  Google Scholar
  27. Peterson DS, Rohr T, Svec F, Fréchet JMJ (2002) Enzymatic microreactor-on-a-Chip: Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem 74: 4081- 4088. » CrossRef   » PubMed   »  Google Scholar
  28. Pramanik NB, Mirza UA, Ning YH, Liu YH, Bartner PL, et al. (2002) Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes. Protein Sci 11: 2677-2687. » CrossRef   » PubMed   »  Google Scholar
  29. Qiao L, Liu Y, Hudson SP, Yang PY, Liu BH (2008) A nanoporous reactor for efficient proteolysis. Chem Eur J 14: 151 - 157. » CrossRef   » PubMed   »  Google Scholar
  30. Sun W, Gao SJ, Wang LJ, Chen Y, Wu SZ, et al. (2006) Microwave-assisted protein preparation and enzymatic digestion in proteomics. Mol Cell Proteomics 5: 769-776. » CrossRef   » PubMed   »  Google Scholar
  31. Svec F (2006) Less common applications of monoliths: Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports. Electrophoresis 27: 947-961. » CrossRef   » PubMed   »  Google Scholar
  32. Tischer W, Wedekind F (1999) Immobilized Enzymes: Methods and Applications. Topic Curr Chem 200: 95 - 126. » CrossRef  »  Google Scholar
  33. Xu XQ, Deng CH, Gao MX, Yu WJ, Yang PY, et al. (2006) Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Adv Mater 18: 3289-3293. » CrossRef  »  Google Scholar
  34. Washburn MP, Wolters DA, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19: 242- 247. » CrossRef   » PubMed   »  Google Scholar
  35. Wolters DA, Washburn MP, Yates JR (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73: 5683-5690. » CrossRef   » PubMed   »  Google Scholar
  36. Zhang YH, Liu Y, Kong JL, Yang PY, Tang Y, et al. (2006) Efficient proteolysis system: A nanozeolite-derived microreactor. Small 2: 1170 - 1173. » CrossRef   » PubMed   »  Google Scholar
  37. Zhu H, Bilgin M, Snyder M (2003) Proteomics. Annu Rev Biochem 72: 783-812. » CrossRef   » PubMed   »  Google Scholar
  38. Zhan XQ, Desiderio DM (2006) Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry. Anal Biochem 354 : 279-289. » CrossRef   » PubMed   »  Google Scholar
Citation: Dawei Q, Yonghui D, Yingchao L, Huaqing L, Chunhui D, et al. (2008) Development of Core-shell Magnetic Mesoporous SiO2 Microspheres for the Immobilization of Trypsin for Fast Protein Digestion. J Proteomics Bioinform 1: 346-358.

Copyright: © 2008 Dawei Q, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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