Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity

A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (microRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by in-solution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.     

Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping

Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-microm-i.d. capillary. This capillary with a pulled 9-12-microm needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20-mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (microSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 microL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.     

Characterization of reaction dynamics in a trypsin-modified capillary microreactor

Application of mild vibration to an immobilized trypsin capillary microreactor can enhance digestion rates for many globular and glycosylated proteins (12-70-kDa range) without additional sample handling. A sinusoid wave form generator and a simple piezoelectric transducer were used to apply vibration in a wide frequency range to the 50-μm-i.d. enzyme microreactor over its entire length. The mass transport properties of the microreactor were quantitatively examined for protein digestions through the use of an artificial globular protein. This was synthesized by covering the surface of 35-nm-diameter latex beads with a peptide (Leu-Arg-Leu). Capillary electrophoresis analysis of the microreactor products showed there were no mass transport-related effects for vibration of the capillary. Digestions of a range of globular protein structures were performed and the products analyzed by capillary electrophoresis. The rate enhancements were found to be related to the stability of the protein tertiary and secondary structure. Cytochrome c showed a dramatic acceleration in rate of digestion as the vibration frequency increased over a range of 200 Hz to 7.1 kHz. The ability to enhance reaction rates for very stable proteins can be gained by additional means of destabilizing the protein, as shown by removal of calcium from α-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical modification to completely denature proteins usually requires considerable sample handling.     

Monolithic bioreactor immobilizing trypsin for high-throughput analysis

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.     

Stable microstructured network for protein patterning on a plastic microfluidic channel: strategy and characterization of on-chip enzyme microreactors

Chemical modification of a poly(methyl methacrylate) (PMMA) microchannel surface has been explored to functionalize microfluidic chip systems. A craft copolymer was designed and synthesized to introduce the silane functional groups onto the plastic surface first. Furthermore, it has been found that, through a silicon-oxygen-silicon bridge that formed by tethering to these functional groups, a stable patterning network of gel matrix could be achieved. Thus, anchorage of proteins could be realized onto the hydrophobic PMMA microchannels with bioactivity preserved as far as possible. The protein homogeneous patterning in a microfluidic channel has been demonstrated by performing microchip capillary electrophoresis with laser-induced fluorescence detection and confocal fluorescence microscopy. To investigate the bioactivity of enzymes entrapped within stable silica gel-derived microchannels, the suggested scheme was employed to the construction of immobilized enzyme microreactor-on-a-chip. The proteolytic activity of immobilized trypsin has been demonstrated with the digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL/min, which affords the short residence time less than 5 s. The digestion products were characterized using MALDI-TOF MS with sequence coverage of 75 and 31% observed, respectively. This research exhibited a simple but effective strategy of plastic microchip surface modification for protein immobilization in biological and proteomic research.     

Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.     

Development of trypsin biosensor based on ion sensitive field-effect transistors for proteins determination

This new biosensor for protein determination is based on a possibility of translation of either a proton or hydroxyl ion arising during protein hydrolysis in the presence of trypsin by means of ion selective field effect transistor (ISFET). The conditions of trypsin immobilization and main biosensor characteristics were optimized using hydrolysis reaction of nalpha-benzoyl-l-arginine ethyl ester hydrochloride (BAEE). The trypsin was immobilized on the ISFET surface using a co-reticulation process between the enzyme and BSA (4% trypsin, 6% BSA) in saturated glutaraldehyde vapour. The limit of detection of BAEE with this biosensor is 0.5 mM with a linear dynamics range from 0.5 to 5 mM, and sensitivity is about 6 mV/mM. The time of biosensor response was 5–7 min.The possibility of the application of developed biosensor for detection of small penta-peptide, which is used in some cosmetic products, has been demonstrated.     

Enzyme inhibitor screening by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.     

Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices

Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties of the monoliths lead to very low back pressures enabling the use of simple mechanical pumping to carry out both the immobilization of the enzyme from its solution and the subsequent analyses of substrate solutions. The Michealis-Menten kinetic characteristics of the reactors were probed using a low molecular weight substrate: N-alpha-benzoyl-L-arginine ethyl ester. The effects of immobilization variables such as the concentration of trypsin in solution and percentage of azlactone functionalities in the monolith, as well as the effect of reaction time on the enzymatic activity, and of process variables such as substrate flow velocity and residence time in the reactor, were studied in detail. The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate. The excellent performance of the monolithic microreactor was also demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min, which affords a residence time of only 11.7 s. The digest was then characterized using MALDI-TOF MS, and 102 out of 153 possible peptide fragments were identified giving a sequence coverage of 67%.     

CE-microreactor-CE-MS/MS for protein analysis

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome c and myoglobin generated Mascot MOWSE scores of 107 for cytochrome c and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.     

Capillary monolithic bioreactor of immobilized snake venom phosphodiesterase for mass spectrometry based oligodeoxynucleotide sequencing

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.     

Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.     

Facile Preparation of SiO(2)/TiO(2) Composite Monolithic Capillary Column and Its Application in Enrichment of Phosphopeptides

A novel SiO(2)/TiO(2) composite monolithic capillary column was prepared by sol-gel technology and successfully applied to enrich phosphopeptides as a metal oxide affinity chromatography (MOAC) material. For the monolith preparation, tetramethoxysilane (TMOS) and tetrabutoxytitanium (TBOT) were used as silica and titania source, respectively, and glycerol was introduced to attenuate the activity of titanium precursor, which provided a mild synthetic condition. The prepared monolith was characterized by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results revealed an approximate 1/2 molar ratio of titanium to silica as well as an atom-scale homogeneity in the framework. The scanning electron microscopy (SEM) results demonstrated an excellent anchorage between the column and the inner capillary wall, and nitrogen adsorption-desorption experiments showed a bimodal porosity with a narrow mesopore distribution around 3.6 nm. The prepared monolith was then applied for selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA) as well as human blood serum, nonfat milk, and egg white using an in-tube solid phase microextraction (SPME) system. Our results showed that SiO(2)/TiO(2) composite monolithic capillary column could efficiently enrich the phosphopeptides from complex matrixes. To the best of our knowledge, this is the first attempt for preparing the silica-metal composite monolithic capillary column, which offers the promising application of the monolith on phosphoproteomics study.     

Membrane-based nanoscale proteolytic reactor enabling protein digestion,peptide separation,and protein identification using mass spectrometry

A miniaturized trypsin membrane reactor housed inside a commonly used capillary fitting is developed and demonstrated for enabling rapid and sensitive protein identification by on-line proteolytic digestion and analysis of protein digests using nano-ESI-MS and MALDI-MS. The design and assembly of the capillary fitting-based trypsin membrane reactor are straightforward and highly robust, without the need for expensive fabrication technology and procedures. The resultant protein digests can also be further concentrated and resolved using capillary reversed-phase liquid chromatography or transient capillary isotachophoresis/zone electrophoresis prior to the mass spectrometric analysis in an integrated platform. By comparing these results with the results obtained from our previous studies using plastic microfluidics (Gao et al., Anal. Chem. 2001, 73, 2648-2655), significant reduction in dead volume and sample consumption can be achieved using this newly developed tryptic digestion station. This nanoscale reaction system enables rapid proteolytic digestion in seconds instead of hours for a protein concentration of less than 10(-8) M, consumes very little sample (< or = 5 fmol), and offers capillary interfaces with various separation and mass spectrometry techniques. The ultrafast enzymatic turnover for attaining complete peptide coverage in protein identification is contributed by the highly porous structure of the membrane media, providing excessive trypsin loading while eliminating the constraints of diffusion-limited reaction kinetics.     

Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes

A porous zwitterionic monolith was prepared by thermal copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine and ethylene dimethacrylate inside a 100-mum-i.d. capillary. The resulting monolith was evaluated as a hydrophilic liquid chromatography (HILIC) stationary phase. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical HILIC mechanism was observed at higher organic solvent content (ACN% > 60%). The poly(SPE-co-EDMA) monolith showed very good selectivity for neutral, basic, and acidic polar analytes. For charged analytes, both hydrophilic interactions and electrostatic interactions contributed to their retention, which provide chromatographers more choice to optimize the separations.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot

To increase the throughput of protein identification and characterization in proteome studies, we investigated three methods of performing protein digestion in parallel. The first, which we term "one-step digestion-transfer" (OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight proteins. The second method is based on "in-gel" digestion of all proteins in parallel and termed "parallel in-gel digestion" (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high molecular weight proteins (> 40,000) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60,000) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low molecular weight polypeptides. Peptides liberated during transblotting of proteins through the immobilized trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode.     

Enhanced protein digestion through the confinement of nanozeolite-assembled microchip reactors

An on-chip microreactor was proposed toward the acceleration of protein digestion through the construction of a nanozeolite-assembled network. The nanozeolite microstructure was assembled using a layer-by-layer technique based on poly(diallyldimethylammonium chloride) and zeolite nanocrystals. The adsorption of trypsin in the nanozeolite network was theoretically studied based on the Langmuir adsorption isotherm model. It was found that the controlled trypsin-containing nanozeolite networks assembled within a microchannel could act as a stationary phase with a large surface-to-volume ratio for the highly efficient proteolysis of both proteins at low levels and with complex extracts. The maximum proteolytic rate of the adsorbed trypsin was measured to be 350 mM min-1 microg-1, much faster than that in solution. Moreover, due the large surface-to-volume ratio and biocompatible microenvironment provided by the nanozeolite-assembled films as well as the microfluidic confinement effect, the low-level proteins down to 16 fmol per analysis were confidently identified using the as-prepared microreactor within a very short residence time coupled to matrix-assisted laser desorption-time-of-flight mass spectrometry. The on-chip approach was further demonstrated in the identification of the complex extracts from mouse macrophages integrated with two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry. This microchip reactor is promising for the development of a facile means for protein identification.     

Aptamer-modified monolithic capillary chromatography for protein separation and detection

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation.     

Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres

A fast and efficient proteolysis approach of microwave-assisted protein digestion was developed by using trypsin-immobilized magnetic silica (MS) microspheres. In the work, immobilization of the enzyme onto MS microspheres was very simple and only through a one-step reaction with 3-glycidoxypropyltrimethoxysilane (GLYMO) which provides the epoxy group as a reactive spacer. Considering that the magnetic particles are excellent microwave absorbers, we developed a novel microwave-assisted digestion method based on the easily prepared trypsin-immobilized MS microspheres. This novel digestion method combined the advantages of immobilized trypsin and the rapid-fashion of microwave-assisted digestion, which resulted in high digestion efficiency. BSA and myoglobin were used as model proteins to optimize the conditions of this method. Peptide fragments produced in 15 s could be confidently identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Equivalent or better digestion efficiency was observed comparing to current in-solution digestion. Besides, because of the unique magnetic responsivity, the immobilized trypsin can be isolated easily with the help of an external magnet and thus used repeatedly. High activity was obtained even after seven runs of the trypsin-immobilized MS microspheres. To further verify its efficiency in proteome analysis, one reversed-phase liquid chromatography (RPLC) fraction of rat liver extract was applied. After 15 s incubation, 16 totally unique peptides corresponding to two proteins were identified. Finally, the rat liver sample was used to evaluate its worth for the application. With analysis by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS), comparable digestion efficiency was observed with typical in-solution digestion but the incubation time was largely shortened. This new microwave-assisted digestion method will hasten the application of the proteome technique to biomedical and clinical research.