Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

Calleri, Enrica; Temporini, Caterina; Perani, Eleonora; Stella, Cinzia; Rudaz, Serge; Lubda, Dieter; Mellerio, G.; Veuthey, Jean‐Luc; Caccialanza, Gabriele; Massolini, Gabriella

doi:10.1016/j.chroma.2004.06.034

Cited by 112 publications

(108 citation statements)

References 42 publications

(46 reference statements)

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“…As shown in Table 1, identical sequence coverages of BSA (26%) were obtained before and after renewal, indicating the good regenerability of IMER. Of note is that slightly different peptide sequences might be yielded due to the possible variance in proteolytic digestion for large proteins with multiple cleavage sites, as observed in some previous work [10,12,15,17]. To further evaluate the applicability of IMER for complex sample analysis, $5 mg of proteins from rat liver extract was digested by such an IMER with a residence time of $150 s; the digests were analyzed by nanoRPLC-ESI-MS/MS (with details shown in Supporting Information).…”

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confidence: 87%

“…The walls of bare capillaries and channels [7,8], micro-/nanoparticles or beads [9,10], organic polymer monoliths [11][12][13], and silica monoliths [14][15][16] have been exploited for enzyme immobilization, largely quicken the protein digestion process. Recently, we exploited another kind of material, organic-inorganic hybrid silica monolith, for trypsin immobilization via covalent bonding [17,18].…”

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confidence: 99%

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Efficient proteolysis using a regenerable metal‐ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith

Hou

Liang

et al. 2011

Proteomics

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A metal-ion chelate immobilized enzyme reactor (IMER) supported on organic-inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu 21 , trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed-phase liquid chromatography with ESI-MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in-solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu 21 via EDTA followed by trypsin immobilization with fresh Cu 21 introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For $5 mg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in-solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high-throughput proteome profiling. Keywords:Immobilized enzyme reactor / Metal-ion chelation / Organic-inorganic hybrid silica monolith / Protein digestion / Technology / TrypsinThe fast developing proteomic profiling study has considerably increased the need of multidisciplinary efforts involving sample preparation, protein or peptide separation, mass spectrometry identification, and data processing [1]. To obtain the comprehensive information about proteomes, proteolysis is indispensable for the shotgun strategy, one of the widely adopted approaches in current proteomics workflows [2]. Generally, proteolysis can be accomplished by in-solution digestion, in-gel digestion, or on-column digestion [3]. Among them, on-column digestion, which is achieved by immobilized enzyme reactors (IMERs), has drawn much attention recently, due to the increased Abbreviations: GLYCO, glycidoxypropyltrimethoxysilane; IDA, iminodiacetic acid; IMER, immobilized enzyme reactor; TEOS, tetraethoxysilane Ã Current address:

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confidence: 87%

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confidence: 99%

Efficient proteolysis using a regenerable metal‐ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith

Hou

Liang

et al. 2011

Proteomics

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“…[58][59][60][61] The silicabased monolithic column material showed good chromatographic properties of dual pore size distribution; large macropores and thin, highly porous skeleton. In addition, this material offers high permeability, high mechanical strength, and good organic solvent tolerance.…”

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confidence: 99%

Protein Analysis Using a Combination of an Online Monolithic Trypsin Immobilized Enzyme Reactor and Collisionally-Activated Dissociation/Electron Transfer Dissociation Dual Tandem Mass Spectrometry

Hwang¹,

Cho²,

Kim³

et al. 2012

Bulletin of the Korean Chemical Society

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We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ≥ 3+. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

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“…2,19 A segunda, engloba os biorreatores desenvolvidos para triagem de substratos e para os estudos das características cinéticas das enzimas. 20 Nessa segunda classe, os IMER têm sido amplamente utilizados para separação e identificação de metabólitos, 21 nos estudos de metabolismo dos fármacos, 22,23 para análise e síntese enantiosseletiva, 2,24-26 na digestão proteolítica de proteínas on-line, 27 no controle biotecnoló-gico de fármacos, 28 e para a identificação de substratos e/ou inibidores, na busca por novos ligantes. [29][30][31][32][33][34][35] Também merece destaque o uso de enzimas imobilizadas como biossensores em aplicações biomédicas 36 ou como órgãos artificiais.…”

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Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas

Cardoso¹,

Moraes²,

Cass³

2009

Quím. Nova

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Recebido em 5/10/07; aceito em 11/7/08; publicado na web em 2/12/08 IMMOBILIZATION OF THE ENZYMES ON CHROMATOGRAPHIC SUPPORTS: A TOOL TO RESEARCH OF INHIBITOR COMPOUNDS. The development and characterization of bioreactors or IMER (immobilized enzyme reactors) as research tools are important in the scope of medicinal chemistry and constitute an alternative for the rational development of drugs. This approach does not require highly purified enzymes or a great amount of protein, but increase the enzymatic stability against heat, organic solvents and pH, without too much loss of catalyst activity. Immobilized enzyme reactors (IMER) can be used for the accomplishment of high efficiency screening on-line and, thus inhibitors can be quickly identified. Here, we emphasize the development of IMER by use of different methods of immobilization and chromatographic supports. Their applications, in different areas of research, are also fully discussed.

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Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

Cited by 112 publications

References 42 publications

Efficient proteolysis using a regenerable metal‐ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith

Efficient proteolysis using a regenerable metal‐ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith

Protein Analysis Using a Combination of an Online Monolithic Trypsin Immobilized Enzyme Reactor and Collisionally-Activated Dissociation/Electron Transfer Dissociation Dual Tandem Mass Spectrometry

Imobilização de enzimas em suportes cromatográficos: uma ferramenta na busca por substâncias bioativas

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