Protein tryptic digests analyzed by carrier ampholyte‐based capillary electrophoresis coupled to ESI‐MS

Busnel, Jean‐Marc; Descroix, Stéphanie; Saux, Thomas Le; Terabe, Shigeru; Hennion, Marie‐Claire; Peltre, Gabriel

doi:10.1002/elps.200500808

Cited by 25 publications

(14 citation statements)

References 27 publications

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“…As a consequence, NC have very low conductivity and very low ionic strength while maintaining a satisfying buffering capacity [28]. These properties make them excellent BGEs for CZE [29][30][31][32]. We have also previously shown that added to commercial carrier ampholyte mixtures they could improve CIEF resolution [33].…”

Section: Introductionmentioning

confidence: 92%

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

2008

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The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.

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Section: Introductionmentioning

confidence: 92%

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

2008

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“…25 In this situation, the bound cysteine residues had little influence on Ag NCs. However, when trypsin was introduced, it digested Cyt c to small peptides, 26 and released free cysteine residues, which could decrease the fluorescence intensity of Ag NCs. 27 This explains why the fluorescence intensity could not restore to its initial value.…”

Section: Fluorescence Assay For Trypsinmentioning

confidence: 99%

A Label-free Fluorescence Assay for Trypsin Based on the Electron Transfer between Oligonucleotide-stabilized Ag Nanoclusters and Cytochrome c

Hong

Han

et al. 2014

ANAL. SCI.

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Herein we attempt to develop a simple, specific and costeffective fluorescent method for trypsin activity assay through the combination of Cyt c and Ag NCs. The assay principle is elaborated in Scheme 1. We firstly synthesize oligonucleotide-2014 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. A label-free fluorescent assay for the detection of trypsin by using oligonucleotide-templated silver nanoclusters (Ag NCs) and cytochrome c (Cyt c) has been demonstrated. When negatively charged Ag NCs and positively charged Cyt c are mixed, they tend to form a hybrid complex, and then lead the fluorescence of Ag NCs to be quenched significantly due to electron transfer between Ag NCs and the heme cofactor of Cyt c. In the presence of trypsin, it catalyzes the hydrolytic cleavage of Cyt c to small peptide fragments, and releases the heme moiety from the Ag NCs/Cyt c complex; the quenched fluorescence restores therewith. By virtue of this specific response, the fluorescent biosensor has a linear range of from 0.7 to 4 μg mL -1 and from 9 to 120 μg mL -1 with a detection limit of 58.7 ng mL -1. Aside from the easy manufacture aspect, our method also possesses a high signal-to-background ratio (~11), excellent selectivity and good biocompatibility, which makes it a promising bioanalysis for a trypsin activity assay. Keywords

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“…In this last case, ultranarrow pH cuts (encompassing typically 0.1 pH units) are used, thus the method is implemented not as a focusing technique, but in a zone electrophoretic approach in a capillary format (CZE) [14][15][16]. Interestingly, this new methodology is exploited not only for proteins, but for peptide separations as well, by interfacing the capillary outlet directly with an MS instrument [17].…”

Section: Introductionmentioning

confidence: 99%

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9–11 interval

2007

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As a last part of an investigation on all 2-pH-unit intervals of carrier ampholytes (CAs) for IEF (see Electrophoresis 2006, 27, 3919-3934; 2006, 27, 4849-4858; 2007, 28, 715-723) two different lots of Servalyt CAs, in the pH 9-11 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by capillary electrophoresis mass-spectrometry of 10 out of 20 fractions harvested, in the second dimension. The findings: the two lots contain 65 and 69 different M(r) compounds, in the M(r) interval of 232-667 Da, for a total of 341-387 isoforms, respectively. Since this is a chaotic organic synthesis, the high reproducibility (here demonstrated for the first time during the 40 years of existence of CAs) of the synthetic process (for two batches produced at 6 years of distance) is remarkable, considering that a 94% agreement for the individual chemicals and 88% agreement for the total number of isoforms for the two lots is found. It is additionally demonstrated that the lower pI species are accompanied by considerably more isoforms than the high pI forms and that in all cases such isoforms consist of family of compounds clustered around the pI of the parental form, with a pI spread of ca. 0.1-0.2 pH units.

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Protein tryptic digests analyzed by carrier ampholyte‐based capillary electrophoresis coupled to ESI‐MS

Cited by 25 publications

References 27 publications

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

A Label-free Fluorescence Assay for Trypsin Based on the Electron Transfer between Oligonucleotide-stabilized Ag Nanoclusters and Cytochrome c

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9–11 interval

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