Optimization of Online Peptide Mapping by Capillary Zone Electrophoresis

Licklider, Larry; Kuhr, Werner G.

doi:10.1021/ac00096a003

Cited by 56 publications

(39 citation statements)

References 19 publications

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“…The same coupled capillary CZE instrument as previously described by Kuhr [53] was used for further experiments and three microreactors were prepared with two proteases (trypsin and pepsin) and a peptidase, carboxypeptidase-Y [55]. These proteolytic enzymes are distinguished by the large differences in their specificities toward peptide bond cleavages and their pH of greatest catalytic efficiency.…”

Section: Microreactor Coupled With Separation Capillarymentioning

confidence: 99%

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

Massolini¹,

Calleri

2005

J. Sep. Science

151

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The ability to rapidly and efficiently digest and identify an unknown protein is of great utility for proteome studies. Identification of proteins via peptide mapping is generally accomplished through proteolytic digestion with enzymes such as trypsin. Limitations of this approach consist in manual sample manipulation steps and extended reaction times for proteolytic digestion. The use of immobilized trypsin for cleavage of proteins is advantageous in comparison with application of its soluble form. Enzymes can be immobilized on different supports and used in flow systems such as immobilized enzyme reactors (IMERs). This review reports applications of immobilized trypsin reactors in which the IMER has been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to MS analysis. Immobilization procedures including supports, mode of integration into separation systems, and methods are described.

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Section: Microreactor Coupled With Separation Capillarymentioning

confidence: 99%

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

Massolini¹,

Calleri

2005

J. Sep. Science

151

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“…12±24 This approach affords advantages over conventional solution digestion in that it minimizes sample loss during handling and transfer, reduces the reaction time required, decreases autolysis since the protease is immobilized on stationary support, and, most importantly, improves protease cleavage efficiency and yields relatively reproducible digestion fragments. 13,15,16,18,19,25 However, this tech-nique has so far largely been used to characterize commercially available protein standards. In this report, on-line proteolytic digestion LC/MS was applied to study specific drug-protein interactions, namely MeDTC-SO, the putative active metabolite of disulfiram, and recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH).…”

Section: ±11mentioning

confidence: 99%

Identification of the protein-drug adduct formed between aldehyde dehydrogenase andS-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

Shen

Johnson

Mays

et al. 2000

Rapid Commun. Mass Spectrom.

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Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

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“…Several research groups have described enzyme reactors coupled to HPLC [13,20] or CE [6][7][8]21]. Recent applications include digestion of the proteins separated using HPLC [14] or 2-D PAGE [22].…”

Section: Introductionmentioning

confidence: 99%

Chararacterization of a monolithic immobilized trypsin microreactor with on‐line coupling to ESI‐MS

Křenková

Bı́lková

Foret³

2005

J of Separation Science

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The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 lm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one-or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto-to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 258C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 378C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.

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Optimization of Online Peptide Mapping by Capillary Zone Electrophoresis

Cited by 56 publications

References 19 publications

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

Identification of the protein-drug adduct formed between aldehyde dehydrogenase andS-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

Chararacterization of a monolithic immobilized trypsin microreactor with on‐line coupling to ESI‐MS

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