Mass spectrometric analysis of maleimide CyDye labelled model peptides

Wheeler, Jun X.; Wait, Robin; Stone, Timothy; Wootton, Lucie; Lewis, S. Judd; Fowler, Susan J.; Cummins, W. Jon

doi:10.1002/rcm.1220

Cited by 8 publications

(7 citation statements)

References 12 publications

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“…To address the suitability for MS identification, the maleimide DIGE dyes have been shown to be compatible with MS analyses -with both modified and unmodified model peptides from a labeling mixture (dye: thiol 2:1) appearing in a MALDI scan [16] -and we, as well, have previously demonstrated the suitability of the BODIPY dyes for MALDI MS [11].…”

Section: Discussionmentioning

confidence: 99%

“…This may raise a concern that the DIGE saturation protocol is not truly saturating, thus impacting its accuracy and reproducibility. A second issue is that Cy3 and Cy5 are charged but neutral by virtue of their quaternary amine (pK a > 12) and sulfonic acid (pK a < 1) moieties [16]. Thus, their impact on the pI of modified proteins will depend on the degree of substitution but will at the least compress the IEF separation profiles on a 2D gel, making acidic proteins more basic and basic proteins more acidic.…”

Section: Discussionmentioning

confidence: 99%

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Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

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We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed.We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

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“…The addition of another independent detection method could be useful to facilitate detection and quantification of peptides and proteins by LC-MS. The examples of detection of biomolecules using HPLC-MS in combination with fluorescence detection were reported in the literature [15][16][17][18][19][20][21]. These studies showed that the analyses of fluorescent biomolecules by LS-ESI-MS and LC-MALDI-MS are feasible.…”

Section: Introductionmentioning

confidence: 95%

Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Saraswat

Snyder

Isailović

2012

Journal of Chromatography B

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“…One study suggested that labelled peptides appear to be less efficiently detected by MS, particularly in MS/MS mode, than their unlabelled counterparts 29. An application note suggests that the labelled and unlabelled species can both be detected by MS, however, and that the presence of the sulphonate group on the maleimide dyes does not impair fragmentation 30. In any case, the putative discrepancy did not appear to limit protein identification, as there were generally sufficient unlabelled peptides for MS to proceed normally 29.…”

Section: Protein Identification From Saturation Dige Gels: Automatementioning

confidence: 99%