Online Microreactors/Capillary Electrophoresis/Mass Spectrometry for the Analysis of Proteins and Peptides

Licklider, Larry; Kuhr, Werner G.; Lacey, Martin P.; Keough, T.; Purdon, Michael P.; Takigiku, Ray

doi:10.1021/ac00118a021

Cited by 96 publications

(67 citation statements)

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“…On the contrary, the immobilized enzyme has been developed for characterization of proteins with benefits from the reusability and stability of enzyme, the higher digestion efficiency of analyte proteins, and no autolysis products [4][5][6][7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…But the bioreactive probes are critical to reuse because of the loss of the enzymatic activity due to the rigorous washing tip process or the occupation of the active site of the proteases by the excessive matrix. Most reported microreactors are based on the format of microcolumns, that is, the enzyme is immobilized on support media [6,7], and then packed into the microcolumn, or onto the surface of silicon-based microfluidic chips [8,9] or on the fused-silica capillary directly by the covalent bonding method [10][11][12]. Compared with digestion of protein by the immobilized enzyme on probe tips, less analyte is needed for the mass mapping and the enzyme is reusable.…”

Section: Introductionmentioning

confidence: 99%

“…Compared with digestion of protein by the immobilized enzyme on probe tips, less analyte is needed for the mass mapping and the enzyme is reusable. Licklider et al [10] reported the use of a capillary microreactor for protein digestion, followed by online ESI and off-line MALDI analysis. The microreactor is vibrated with an electric engraving tool to enhance reaction rates and reduce the digestion time to about 30 min [11].…”

Section: Introductionmentioning

confidence: 99%

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Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

Guo

Lei

et al. 2003

Electrophoresis

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Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometryPeptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH 4 HF 2 . The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 507C, even 20 fmol cytochrome c could be well digested and detected.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

Guo

Lei

et al. 2003

Electrophoresis

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“…In both approaches, to obtain detailed structural information, proteins are selectively cleaved into smaller polypeptide fragments by controlled chemical or enzymatic reactions Wolters et al, 2001;Zhu et al, 2003 (Nalivaeva and Turner, 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; Ekstrom 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.…”

Section: Abstract: Mesoporous Sio 2 Microspheres ; Peptide Mapping Anmentioning

confidence: 99%

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

Qi¹,

Deng²,

Liu³

et al. 2008

J Proteomics Bioinform

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In the work, we developed glycidoxypropyltrimethoxysilane (GLYMO)-modified Fe O @SiO core and perpendicularly aligned mesoporous SiO 2 shell (designated Fe O @nSiO @mSiO 2 ) as the novel substrate for the immobilization of large amount of trypsin and applied it for fast protein digestion.Firstly, Fe 3 O 4 @nSiO 2 @mSiO 2 microspheres were synthesized. Then, the surface of the microspheres was functionalized with GLYMO for enzyme immobilization.The amount of trypsin immobilized on was about 188 g/mg, which was much more than that on the previous magnetic materials. Using the trypsin-immobilized magnetic mesoporous SiO 2 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 SiO 2 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 SiO 2 microspheres ; Peptide mapping analysis ; Microwave-assisted digestion ; MALDI-TOF MS IntroductionProteomic 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 Wolters et al., 2001;Zhu et al., 2003 (Nalivaeva and Turner, 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; Ekstrom 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 (Kvenková et al., 2005). And in many other reported approaches (Kvenková et al., 2005;Luo et al., 2002), the epoxide functional groups for enzyme immobilization are applied. As the auth...

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“…[39][40][41][42][43][44][45][46] Com este propósito, diferentes configurações de interfaces já foram desenvolvidas, tal como acoplamento em linha com MALDI-MS. [47][48][49] Jin et al 50 apresentaram um trabalho revolucionário construindo um sistema de reator enzimático em linha no qual a bomba seringa, tradicionalmente usada para gerar o fluxo hidrodinâmico, foi substituída pelo fluxo eletroosmótico, levando as proteínas até o reator enzimático. As enzimas foram imobilizadas em uma fase estacionária e neste sistema foi possível, em uma única etapa, promover a digestão de proteínas em 12 min com um sistema integrado de eletroforese na forma de um chip e sua caracterização em um sistema MALDI-TOF-TOF (time of flight).…”

Section: Introductionunclassified

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

Assunção¹,

Bechara²,

Simionato³

et al. 2008

Quím. Nova

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Recebido em 29/1/08; aceito em 7/5/08; publicado na web em 8/10/08 CAPILLARY ELECTROPHORESIS COUPLED TO MASS SPECTROMETRY (CE-MS): TWENTY YEARS OF DEVELOPMENT. CE-MS has been increasingly used for analysis of a vast array of compounds. This article reviews the different electrophoretic modes, interfaces and mass analyzers that are commonly used in the CE-MS coupling, as well as the technique advantages and performance characteristics. A large compilation of CE-MS applications is also presented. Therefore, this review is both a guide for beginners and a collection of key references for people who are familiar to the technique. Furthermore, this is the first CE-MS review published in a Brazilian journal and marks the installation of the first two commercial CE-MS units in Sao Paulo State.Keywords: capillary electrophoresis; electrospray ionization; mass spectrometry. introdUÇÃoA eletroforese capilar (CE) vem ocupando um espaço crescente na química analítica como uma poderosa ferramenta de separação e identificação de uma ampla gama de moléculas. A CE possui alta resolução e eficiência, parâmetros estes de grande importância em um processo de separação. Outra vantagem é o baixo custo operacional: mesmo em condições em que se empregam solventes orgânicos, os volumes consumidos de amostra e eletrólito são desprezíveis comparados à cromatografia líquida de alta eficiência (HPLC), até mesmo quando a última é utilizada em escala capilar. Ressaltamos também que os processos eletroforéticos têm como característica positiva a pequena quantidade de amostra injetada (ordem de nL), fator de importância em aplicações clínicas. Porém a maior contribuição que a CE oferece é sua complementaridade aos métodos já estabelecidos, devido à variedade de modos de separação existentes -e muitos ainda em desenvolvimento -os quais permitem a análise de praticamente todas as classes de compostos.1-3 A CE acoplada à espectrometria de massas (CE-MS) surgiu, aproximadamente, há duas décadas, e foi e vem sendo usada em diferentes aplicações, com crescente aceitação. O sucesso desta combinação alia o alto poder de resolução e eficiência da CE com a universalidade e sensibilidade que a MS oferece frente a outros sistemas de detecção como, por exemplo, espectrofotometria na região do UV-visível, bem como fluorescência induzida a laser (LIF -laser induced fluorescence), que são pouco informativos em relação à estrutura do analito. Um exemplo de tais características de MS envolve seu uso juntamente a ferramentas computacionais, o que é altamente recomendado em estudos envolvendo amostras complexas (como proteínas, peptídeos e metabólitos), já que informações como massa molecular e estrutura (MS em tandem) são indispensáveis para caracterização e quantificação de tais analitos.Desta forma, as informações oferecidas pela segunda dimensão do detector de massas constituem uma outra vantagem do uso de CE-MS. Assim, solutos que apresentam pequenas variações no tempo de migração, ou até mesmo que migram com a mesma velocidade, podem ser identificados. Nest...

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Online Microreactors/Capillary Electrophoresis/Mass Spectrometry for the Analysis of Proteins and Peptides

Cited by 96 publications

References 0 publications

Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

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

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

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