Acceleration of Microwave-Assisted Enzymatic Digestion Reactions by Magnetite Beads

Chen, Wei Yu; Chen, Yu-Chie

doi:10.1021/ac0614893

Cited by 103 publications

(87 citation statements)

References 25 publications

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“…This approach exploits unique microwave properties to accelerate the enzymatic digestion. In recent years, microwaveassisted proteolytic digestion procedures, including tryptic [6][7][8] and acid-mediated proteolysis [9][10][11][12], have enabled samples to be prepared more rapidly for bottom-up analysis.…”

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confidence: 99%

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Reddy

Hsu

et al. 2010

J. Am. Soc. Mass Spectrom.

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Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60°C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern. (J Am Soc Mass Spectrom 2010, 21, 421-424)

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confidence: 99%

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Reddy

Hsu

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…Centrifuge-assisted tryptic digestion was conducted at 37 o C under various centrifugation speeds (10,000×g, 20,000×g, and 30,000×g) and digestion times (1,2,3,6, and 16 h). The effect of acetonitrile (ACN) on the centrifuge-assisted tryptic Vol.…”

Section: 6mentioning

confidence: 99%

Effect of centrifugation on tryptic protein digestion

Kim¹,

Kim²,

Lee³

et al. 2017

Analytical Science and Technology

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This study investigated the effect of centrifugation on tryptic digestion. This was done by applying different centrifugation speeds (6,000, 8,000, 10,000, 20,000, and 30,000×g) over various durations (0, 10, 20, 30, 40, 50, and 60 min) to digest two model proteins -cytochrome c and myoglobin. The intact proteins and resulting peptides were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Centrifugation greatly improved the tryptic digestion efficiency of cytochrome c, where either an increase in centrifugation speed or in digestion duration significantly improved the digestion of cytochrome c. However, centrifugation did not noticeably improve the digestion of myoglobin; 16 h of centrifuge-assisted tryptic digestion at 30,000×g barely removed the myoglobin protein peak. Similar results were also obtained when using conventional tryptic digestion with gentle mixing. When acetonitrile (ACN) was added to make 10% ACN buffer solutions, the myoglobin protein peak disappeared after 6 h of digestion using both centrifugeassisted and conventional tryptic digestions.

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“…As we know, in many references (Juan et al, 2005;Chen et al, 2007;Fan et al, 2005;Qiao et al, 2008), MYG (MW 16 700) or Cyt-C (MW 12 384) and BSA (MW 66 000) was used as model proteins to test the digestion efficiency. In our work, the three proteins were also used.…”

Section: Microwave-assisted Protein Enzymatic Digestionmentioning

confidence: 99%

“…Pramanik et al also applied microwave technology to digest known proteins in solution or gel with trypsin in 10 min including one protein that was tightly folded and extremely resistant to denaturation (bovine ubiquitin) . More recently, Chen et al, (2007) found that MAPED could be further accelerated by magnetite microspheres, which had been proved to be excellent absorbers of microwave radiation (Chen et al, 2007). Magnetic microspheres possess the unique magnetic responsivity, which means that the magnetic microspheres are not only available as highly dispersed suspensions in a wide range of sizes (50 nm to 5 mm) and permit modification on their surface, but also offer the advantage of straightforward and fast handling with the help of an applied magnetic field.…”

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confidence: 99%

“…Since the magnetic microspheres are excellent microwave absorbers; and thus greatly improve the efficiency of protein digestion. We compared the digestion efficiency of our protocol with other reported methods (Chen et al, 2007;Fan et al, 2005;Zhang et al, 2006;Guo et al, 2003), and the results were listed in Table S2 (see supporting information). The digestion time speared from 20 s to 15 min with different digestion method, and the sequence coverage ranges from 77% to 89% of cytochrome c and 44% to 89% of myglobin.…”

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confidence: 99%

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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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Acceleration of Microwave-Assisted Enzymatic Digestion Reactions by Magnetite Beads

Cited by 103 publications

References 25 publications

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Effect of centrifugation on tryptic protein digestion

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

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