A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications
TL;DR: The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that thesol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use.
Abstract: The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in...
Citations
More filters
TL;DR: The beads were grafted with poly(hydroxylpropyl methacrylate) (pHPMA) and used for immobilization of trypsin via click chemistry and the optimal pH value for the immobilized enzyme was slightly higher compared to free enzyme.
Abstract: In the present work, magnetic poly(2-hydroxyethyl methacrylate–ethyleneglycol dimethacrylate) (mp(HEMA-EGDMA)) beads were prepared by suspension polymerization. The beads were grafted with poly(hydroxylpropyl methacrylate) (pHPMA) and used for immobilization of trypsin via click chemistry. The p(HPMA) units in the brushes made the fibrous polymer hydrophilic. Synthesized beads were characterized by swelling studies, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The bromo groups of the grafted polymer were modified into azide groups, and the enzyme was alkyne-functionalized for conjugation with azide-modified magnetic beads via click reaction. The kinetic constants (Km and Vmax) were determined by measuring initial reaction rates using the artificial chromogenic substrate N-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA). The optimal pH value for the immobilized enzyme was slightly higher compared to free enzyme. The immobilized trypsin possessed much high...
28 citations
TL;DR: The immobilization of enzymes on solid materials is a promising strategy in biotechnological applications and proteomics, and enables a more convenient handling, easy separation from the reaction solution, and cyclic reuse of the enzymes.
Abstract: The immobilization of enzymes on solid materials is a promising strategy in biotechnological applications and proteomics. It can improve the enzymes’ stability, and enables a more convenient handling, easy separation from the reaction solution, and cyclic reuse of the enzymes. In order to investigate the proteolytic properties of a particle-bound protease, chymotrypsin was covalently immobilized on silica and alumina colloidal particles. The enzymatic activity of the bound chymotrypsin at different times, in consecutive proteolytic cycles, and after storage up to several weeks was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Using this approach, the proteolysis products were identified without using artificial protease substrates or intermediate chemicals. Lysozyme was used as a model protein to perform enzymatic digestion using immobilized chymotrypsin and the peptides generated from the proteolytic digestion were determined. Compared to the...
7 citations
Cites background or methods from "A Trypsin Immobilized Sol-Gel for P..."
...Despite the significant attention paid to the development of enzyme immobilization strategies on various supports, a major challenge in this field is the limited availability of reliable methods enabling a direct characterization of enzyme activity and overall enzymatic performance at different operation and storage conditions (Jeng et al. 2007; Lin et al. 2007; Xu et al. 2007; Celikbicak et al. 2013)....
[...]
...…a major challenge in this field is the limited availability of reliable methods enabling a direct characterization of enzyme activity and overall enzymatic performance at different operation and storage conditions (Jeng et al. 2007; Lin et al. 2007; Xu et al. 2007; Celikbicak et al. 2013)....
[...]
Dissertation•
23 Mar 2016
TL;DR: This study prepared glutaraldehydecrosslinked chymotrypsin (GA-CT), which creates insoluble particles, and fabricated immobilized enzyme microreactor (IMER) based on GA-CT, used to digest BSA, myoglobin, a 9-residue peptide and a dipeptide as examples of large, medium and small substrates.
Abstract: Digesting proteins using proteolytic enzymes is a standard method in proteomic studies and bottom-up protein sequencing. Enzymes can be added in solution or gel phase depending on how the protein has been isolated. Immobilized, i.e., insoluble, proteolytic enzymes offer several advantages such as reusability of enzyme, high enzyme-to-substrate ratio, and integration with fluidic systems. In this study, we prepared glutaraldehydecrosslinked chymotrypsin (GA-CT), which creates insoluble particles. The immobilization efficiency was determined by absorbance spectrophotometry and found to be 96% of the total amount of chymotrypsin added. Different immobilization (i.e., crosslinking) conditions such as buffer composition/pH and initial mass of CT during crosslinking as well as different storage conditions such as temperature, time and humidity for the GA-CT particles were evaluated by comparing capillary electrophoretic (CE) peptide maps of protein standards digested with the particles. The GA-CT particles were used to digest BSA as an example of a large folded protein that needs denaturation prior to digestion, and casein-fluorescein isothiocyanate (FITC) as an example of a small, labeled substrate to test enzyme activity in the presence of substrate-bound fluorescent groups. Peptide mapping of digests from GA-CT particles was achieved by CE with ultraviolet (UV) absorbance or laser induced fluorescence (LIF) detection. FITC-labeled casein was digested by GA-CT to the same extent as with free (i.e., soluble) CT. An immobilized enzyme microreactor (IMER) was fabricated by immobilizing CT inside a 250 μm i.d. fused-silica capillary tube pre-treated with 3-aminopropyltriethoxysilane to functionalize the inner walls with amine groups. Glutaraldehyde was reacted with the amine groups and then CT was immobilized by crosslinking to the GA. IMERs based on GA-CT were fabricated using an automated CE system and used to digest BSA, myoglobin, a 9-residue peptide and a dipeptide as examples of large, medium and small substrates. Digests were studied by comparing peptide maps obtained by CE coupled to either UV or mass spectrometric (MS) detection in order to evaluate immobilization conditions as a function of buffer composition/pH and reaction times. A separate study, which used fluorescence microscopy to investigate the extent and location of GA-CT immobilization in the IMER, showed that immobilization
6 citations
Cites background or methods from "A Trypsin Immobilized Sol-Gel for P..."
...Immobilized enzyme reactors, once seen only in process chemistry, are being used more and more frequently in proteomics studies for protein digestion [36, 47, 79, 164]....
[...]
...These can be divided into four main categories [58]: first, binding to a carrier, for example magnetic particles [38, 39] or glass beads and polymers as a support [42-44, 156]; second, encapsulation using a polymer [45]; third, entrapment using sol-gels [46, 47]; and forth, by crosslinking of enzyme molecules [48-53]....
[...]
...These methods include: binding to a solid-phase carrier, [38, 39] or glass beads as a support [40-44]; encapsulating by a polymer [45]; entrapping using sol-gels [46, 47]; and crosslinking as a carrier free immobilized enzyme [48-53]....
[...]
TL;DR: A supportless immobilization strategy that uses glutaraldehyde-mediated crosslinking to render proteolytic enzymes insoluble for facile protein digestion and facilitates the passage of substrate through the reactor.
Abstract: Immobilized enzyme microreactors based on proteases for proteomics studies are usually made from enzyme bound to a solid-phase support such as particles packed into a cartridge. Our group has developed a supportless immobilization strategy that uses glutaraldehyde-mediated crosslinking to render proteolytic enzymes insoluble for facile protein digestion. Although this works well in batch format, in-situ crosslinking within a microcolumn-based enzyme microreactor is less straight-forward. A microreactor was fabricated by immobilizing chymotrypsin, a proteolytic enzyme, inside a 250-µm i.d. fused silica capillary that was first functionalized with amino groups before adding glutaraldehyde and enzyme. The extent and location of enzyme immobilization within the capillary tubing was probed by reacting fluorescein isothiocyanate with residual amino groups in the microreactor and imaging the capillary by confocal laser fluorescence microscopy. The images imply that chymotrypsin immobilization occurred mo...
4 citations
Cites background from "A Trypsin Immobilized Sol-Gel for P..."
...…chemistry, are being used more and more frequently in proteomics studies for protein digestion (Urban, Goodall, and Bruce 2006; Ma et al. 2011; Celikbicak et al. 2014; Safdar, Spross, CONTACT Karen C. Waldron karen.waldron@umontreal.ca Department of Chemistry, Université de Montréal, 6128…...
[...]
05 Jul 2017
TL;DR: Enrichment of (phospho‐ and glyco‐) peptides was successfully applied using standard proteins including β‐casein, fetuin, and IgG as well as some complex medium and sol‐ gel materials were compared with commercial metal oxides regarding their modified peptide enrichment performances.
Abstract: Glycosylation and phosphorylation are two of the most commonly seen and important post‐translational modifications (PTMs) of proteins. Both play key role in many vital bio‐ logical processes. Mass spectrometry is the most favored technique used for monitoring the dynamics of PTMs. Mass spectrometric analysis of phosphopeptide and glycopeptide is a crucial step in phosphoproteomics and glycoproteomics to understand the role of these modifications in the cellular pathways. Enrichment and purification of (phospho‐ and glyco‐) peptides and glycans are recommended prior to mass spectrometric analy‐ sis because of the lower amount of modified peptides in a peptide mixture. Herein, we present titanium/silica and zirconium/silica sol‐gel materials for the enrichment of (phos‐ pho‐ and glyco‐) peptides and glycans to enhance MALDI‐MS analysis performance. Enrichment of (phospho‐ and glyco‐) peptides was successfully applied using standard proteins including β‐casein, fetuin, and IgG as well as some complex medium. The sol‐ gel materials were compared with commercial metal oxides regarding their modified peptide enrichment performances.
3 citations
Cites methods from "A Trypsin Immobilized Sol-Gel for P..."
...Sol‐gel‐based materials have been used for various bioapplications, such as enzyme encapsulation [27], immobilization [28], and biosensor [29]....
[...]
References
More filters
TL;DR: A rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography and tandem mass spectrometry to separate and fragment peptides is described.
Abstract: We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.
2,236 citations
TL;DR: 4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III.
Abstract: 4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III. Proteomes and Post-Translational Modifications 285 A. Proteomes 285 1. The Analytical Challenge 285 2. Analysis of Protein−Protein Complexes 286 B. Post-Translational Modifications 286 1. Background 286 2. Detection and Purification of Phosphoproteins 288
1,416 citations
"A Trypsin Immobilized Sol-Gel for P..." refers methods in this paper
...In the most widely used technique, bottomup proteomics, proteins are initially digested by specific proteolytic enzymes and then the peptide mixture is analyzed using a mass spectrometer (Han, Aslanian, and Yates 2008; Yates, Ruse, and Nakorchevsky 2009)....
[...]
TL;DR: This review dissects the overall framework of the MS experiment into its key components, and highlights both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis.
Abstract: Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2–5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10 3 . High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.
1,006 citations
TL;DR: N nanoparticle-based immobilization of enzymes showed a broader working pH and temperature range and higher thermal stability than the native enzymes, and it is possible that co-immobilization of multi-enzymes could be achieved on these nanoparticles.
968 citations
TL;DR: Although bottom-up proteomics remains the workhorse for proteomic analysis, middle-down and top-down strategies should allow more complete characterization of protein isoforms and post-translational modifications.
661 citations