title: Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes.
abstract: Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye DIGE Cy 3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.
authors.affiliation: Global Protein Science & Supply, Enabling Science and Technology (Biology), AstraZeneca, 13S28 Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Shaw
authors.forename: Rachel
authors.initials: R
authors.lastname: Rowlinson
authors.forename: Janice
authors.initials: J
authors.lastname: Nickson
authors.forename: Tim
authors.initials: T
authors.lastname: Stone
authors.forename: Alison
authors.initials: A
authors.lastname: Sweet
authors.forename: Karen
authors.initials: K
authors.lastname: Williams
authors.forename: Robert
authors.initials: R
authors.lastname: Tonge
mesh_terms: D000818:Animals; D003545:Cysteine / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008027:Light; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008956:Models, Chemical; D011506:Proteins / Q000737:chemistry; D012984:Software; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D003545:Cysteine