title: Quantitative DY-maleimide-based proteomic 2-DE-labeling strategies using human skin proteins.
abstract: Sensitive differential proteomic analysis is challenging and often limited by distinct labeling or tagging strategies. In this study, we have examined the sensitivity, linearity, and photophysical properties of novel protein labeling DY-maleimide dyes (DY-505-MAL, DY-555-MAL and DY-635-MAL). All MS compatible DY-maleimide dyes exhibited excellent emission spectra, high sensitivity, and high linearity, when applied to standard 1-DE protein analysis. Correspondingly, 2-DE analysis of DY-635-MAL or DY-505-MAL maximal-labeled human keratinocyte proteins displayed remarkably high sensitivity. Compared with a standard fluorescent protein stain, DY-635-MAL or DY-505-MAL 2-DE analysis demonstrated equally high spot quality with an overall increase in the number of spots detectable (up to threefold higher;>1000 spots/gel). However, as determined with a FLA-5100 imaging system, comparative MultiGauge, and Delta2D analysis, not all DY-maleimide dyes possessed DIGE compatible fluorescent emission properties. However, DY-505-MAL and DY-635-MAL were found to be suitable for more complex, time and gel intensive, focused multiplexing analyses. Notably - as demonstrated with allergen-stimulated human skin proteins - defined, singular DY-maleimide dye protein labeling (SDPL) allows high quality, time saving, simple, and reliable differential proteomic examination.
authors.affiliation: Research Group for Immunology & Proteomics, Department of Dermatology and University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Dietz
authors.forename: Alberto
authors.initials: A
authors.lastname: Bosque
authors.forename: Patrick
authors.initials: P
authors.lastname: Pankert
authors.forename: Stefanie
authors.initials: S
authors.lastname: Ohnesorge
authors.forename: Patrick
authors.initials: P
authors.lastname: Merz
authors.forename: Alberto
authors.initials: A
authors.lastname: Anel
authors.forename: Martina
authors.initials: M
authors.lastname: Schnölzer
authors.forename: Hermann-Josef
authors.initials: HJ
authors.lastname: Thierse
mesh_terms: D003627:Data Interpretation, Statistical; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry* / Q000378:metabolism; D016014:Linear Models; D008301:Maleimides / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000378:metabolism; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D011506:Proteins; D012709:Serum Albumin