False labelling due to quenching failure of <i>N</i>‐hydroxy‐succinimide–ester‐coupled dyes

Wang, Weiqun; Ackermann, Doreen; Mehlich, Anna-Maria; König, Simone

doi:10.1002/pmic.200900679

Cited by 12 publications

(12 citation statements)

References 23 publications

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“…The concept of adding an ionizable section to a molecule is comparable to labeling analytes with fluorescence tags [24][25][26][27]. This approach is also used for other ionization techniques in mass spectrometry to enhance or enable ionization [9,28,29].…”

Section: Resultsmentioning

confidence: 99%

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New derivatization strategies for the ultrasensitive analysis of non-aromatic analytes with APLI-TOF-MS

2015

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Atmospheric pressure laser ionization (APLI) (Constapel et al. in Rapid Commun Mass Spectrom 19:326-336, 2005; Schiewek et al. in Anal Chem 79:4135-4140, 2007; Schmitz and Benter (2007) J Chromatography Library 72:89-114) is an ultra-sensitive and selective ionization method for the determination of polycyclic aromatic compounds. The selectivity is an advantage but also a disadvantage. To overcome the limitation of APLI to aromatic compounds, we have already reported some derivatization strategies for common organic functional groups (Schiewek et al. in Angew Chem Int Ed 47:9989-9992, 2008). Here, we describe the synthesis of new APLI ionization labels and their use in new derivatization strategies. These new labels widen the range of APLI-(TOF)MS analysis to non-aromatic compounds with the most common functional groups such as amines, alcohols and carboxylic acids to gas chromatography (GC) and lower the limits of detection (LOD) down to concentrations as small as pmol/L and attomole on column.

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Section: Resultsmentioning

confidence: 99%

“…NHS esters (4) are also used for the derivatization of amines. This derivatization is performed under mild conditions and is prone to the presence of water; in fact, it is used for labeling analytes in aqueous solutions [24,29]. Figure 5 shows the analysis of propylamine (250 nM) labeled with 3.…”

Section: Aminesmentioning

confidence: 99%

New derivatization strategies for the ultrasensitive analysis of non-aromatic analytes with APLI-TOF-MS

2015

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“…Building on the earlier experiments [1], we first reversed labelling and quenching. To that end, 400 pmol Cy2, Cy3, and Cy5, respectively, were quenched with 10.000 pmol lysine as advised [2] and then 50 µg protein were added (for experimental design and protocols see Text S1, Table S1 and Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…It was recently shown [1] that quenching of N -hydroxy-succinimide (NHS)-ester-coupled dyes in comparative fluorescence gel electrophoresis may require a major excess of quencher in order to avoid unspecific labelling. As much as 2.500-fold excess of reaction partners needs to be present to silence the activated dye molecules sufficiently.…”

Section: Introductionmentioning

confidence: 99%

“…This is due, first, to continued labelling with the dye assigned to the individual sample, because the labelling time cannot be controlled and, second, to unspecific labelling of the other two samples of the set when all three samples are pooled in preparation for isoelectric focussing (Figure S1). We have shown extensively before [1] that the recommended amount of the quencher lysine is not sufficient in labelling protocols as they are used in DIGE [2]; cross-talk of one proteome (sample 1, e.g. control) in the fluorescent gel image of the second proteome (sample 2, e.g.…”

Section: Introductionmentioning

confidence: 99%

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Impact of Quenching Failure of Cy Dyes in Differential Gel Electrophoresis

et al. 2011

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BackgroundDifferential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated.Methodology/Principal FindingsExperiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye.Conclusions/SignificanceIt could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.

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Comparative fluorescence two‐dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on‐gel generation of a reference protein spot grid

et al. 2012

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The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.

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False labelling due to quenching failure of N‐hydroxy‐succinimide–ester‐coupled dyes

Cited by 12 publications

References 23 publications

New derivatization strategies for the ultrasensitive analysis of non-aromatic analytes with APLI-TOF-MS

New derivatization strategies for the ultrasensitive analysis of non-aromatic analytes with APLI-TOF-MS

Impact of Quenching Failure of Cy Dyes in Differential Gel Electrophoresis

Comparative fluorescence two‐dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on‐gel generation of a reference protein spot grid

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