Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

Seiwert, Bettina; Kärst, Uwe

doi:10.1007/s00216-007-1260-9

Cited by 31 publications

(19 citation statements)

References 39 publications

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“…proteins with on-line electrochemical liquid chromatography (LC) MS and tryptic peptide analysis with MS/MS (Seiwert & Karst, 2007;Seiwert, Hayen, & Karst, 2008). The linkage and formation of disulfide bonds after reduction or oxidation was studied with tagging and MS to determine protein activation mechanisms (Barbirz, Jakob, & Glocker, 2000).…”

Section: Cysteine-tagging Strategiesmentioning

confidence: 99%

Cysteine tagging for MS‐based proteomics

Giron

Dayon

Sanchez

2010

Mass Spectrometry Reviews

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Amino acid-tagging strategies are widespread in proteomics. Because of the central role of mass spectrometry (MS) as a detection technique in protein sciences, the term "mass tagging" was coined to describe the attachment of a label, which serves MS analysis and/or adds analytical value to the measurements. These so-called mass tags can be used for separation, enrichment, detection, and quantitation of peptides and proteins. In this context, cysteine is a frequent target for modifications because the thiol function can react specifically by nucleophilic substitution or addition. Furthermore, cysteines present natural modifications of biological importance and a low occurrence in the proteome that justify the development of strategies to specifically target them in peptides or proteins. In the present review, the mass-tagging methods directed to cysteine residues are comprehensively discussed, and the advantages and drawbacks of these strategies are addressed. Some concrete applications are given to underline the relevance of cysteine-tagging techniques for MS-based proteomics.

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Section: Cysteine-tagging Strategiesmentioning

confidence: 99%

Cysteine tagging for MS‐based proteomics

Giron

Dayon

Sanchez

2010

Mass Spectrometry Reviews

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“…[7][8][9][10][11][12][13][14][15] It has also been reported that an electrochemical reaction coupled on-line to LC-MS is a promising approach in drug metabolism and protein research. [16][17][18][19][20][21][22][23] However, there are few reports on applications to methods for determining organic compounds in biological samples using this technique. 24 The combination of LC-MS/MS and the on-line EC reaction is potentially more selective as a drug quantification method, in biological samples, than ordinary LC-MS/MS.…”

Section: Introductionmentioning

confidence: 99%

Application of On-line Electrochemistry/Electrospray/Tandem Mass Spectrometry to a Quantification Method for the Antipsychotic Drug Zotepine in Human Serum

et al. 2009

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A simple, rapid, and sensitive on-line liquid chromatographic electrochemistry/electrospray/tandem mass spectrometry (LC-EC/ESI-MS/MS) method for the determination of zotepine in human serum was developed using a new generated-electrochemically fragment ion, and was validated. A recent novel technique of LC-EC/ESI-MS/MS that combines LC-MS/MS and the on-line EC reaction is potentially applicable to developing a quantification method for drugs in biological samples. Newly formed products generated by the on-line EC cell are expected to provide appropriate precursor and product ions for the MS/MS determination method. This technique was successfully applied to a drug assay in a biological matrix. After adding imipramine (IS) to a 30-μL aliquot of human serum, the resulting sample was simply deproteinated with acetonitrile for a measurement. The analytical run time was 5 min. The calibration curve was linear in the concentration range of 10 -2000 ng/mL. The intra-assay precision and accuracy were in the range of 1.8 -8.9 and 98.4 -113%, respectively.

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“…The acetonitrile (ACN) for HPLC was obtained from Merck KGaA (Darmstadt, Germany). N-(2-ferrocene-ethyl)maleimide (FEM) was synthesized from ferrocenylmethyltrimethylammonium iodide (obtained from Alfa Aesar, Karlsruhe, Germany) according to a method described in literature [16,17]. Ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) was synthesized in a two-step reaction from ferrocenecarboxylic acid (obtained from Alfa Aesar) according to Karst et al [18,19].…”

Section: Chemicalsmentioning

confidence: 99%

“…Two maleimide-based derivatizing agents are used for this purpose. N-(2-ferrocene-ethyl)maleimide (FEM) was already used for the analysis of proteins and peptides by HPLC/MS [16]. The second reagent, ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) was recently introduced [19] for the detection of low molecular weight aminothiols and was synthesized for the differential labeling approach together with FEM.…”

Section: Differential Derivatization Of Cysteine Residues In Proteinsmentioning

confidence: 99%

“…We describe a new approach of differential protein labeling using two ferrocene-based derivatizing agents. In a recent paper, we reported the development of a LC/MS method based on the derivatization of thiol groups with the derivatizing agent N-(2-ferroceneethyl)maleimide (FEM) [16]. The method presented in this paper uses a two-reagent differential labeling of thiol and disulfide functions in proteins.…”

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confidence: 99%

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Differential labeling of free and disulfide-bound thiol functions in proteins

Seiwert

Hayen

Kärst

2008

J. Am. Soc. Mass Spectrom.

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A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence. (J

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Analysis of cysteine-containing proteins using precolumn derivatization with N-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

Cited by 31 publications

References 39 publications

Cysteine tagging for MS‐based proteomics

Cysteine tagging for MS‐based proteomics

Application of On-line Electrochemistry/Electrospray/Tandem Mass Spectrometry to a Quantification Method for the Antipsychotic Drug Zotepine in Human Serum

Differential labeling of free and disulfide-bound thiol functions in proteins

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