Recent advances in immobilized enzymatic reactors and their applications in proteome analysis

Ma, Junfeng; Zhang, Lihua; Liang, Zhen; Zhang, Weibing; Zhang, Yukui

doi:10.1016/j.aca.2007.08.045

Cited by 110 publications

(78 citation statements)

References 80 publications

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“…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 apparent enzymatic activity and improved digestion efficiency [4][5][6].…”

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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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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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“…It could be also connected with related components directly for on-line digestion [9,10]. Nowadays, a variety of IMERs have been developed with different supporting materials, e.g., membranes [11][12][13], particles [14][15][16], monoliths and so on.…”

Section: Introductionmentioning

confidence: 99%

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

Sun

et al. 2011

Talanta

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“…A typical procedure for cross-linking enzyme involves activation of the primary amine groups of enzyme with cross-linker to create aldehyde groups that can react readily with other primary amine groups of enzymes Ma et al, 2009;. Cross-linking yields depend on the number of the Lys residues of enzyme.…”

Section: Preparation Of Protease-immobilized Microreactorsmentioning

confidence: 99%

“…Enzyme-immobilized www.intechopen.com microreactors have been widely utilized in chemical and biotechnological fields (Liu et al, 2008;Ma et al, 2009;Miyazaki et al, 2008;Asanomi et al, 2011). The protease-immobilized microreactor provides several advantages for proteolysis (Ma et al, 2009); e.g. low degree of auto-digestion even at high protease concentrations and a large surface and interface area that leads to rapid proteolysis.…”

Section: Introductionmentioning

confidence: 99%

“…A microreactor is a suitable reaction system for handling small-volume samples in a microchannel to perform chemical or enzymatic reactions. Enzyme-immobilized www.intechopen.com microreactors have been widely utilized in chemical and biotechnological fields (Liu et al, 2008;Ma et al, 2009;Miyazaki et al, 2008;Asanomi et al, 2011). The protease-immobilized microreactor provides several advantages for proteolysis (Ma et al, 2009); e.g.…”

Section: Introductionmentioning

confidence: 99%

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Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

Yamaguchi¹,

Miyazaki²,

Maeda³

2012

Integrative Proteomics

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Recent advances in immobilized enzymatic reactors and their applications in proteome analysis

Cited by 110 publications

References 80 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

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

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