title: A highly sensitive near-infrared fluorescent detection method to analyze signalling pathways by reverse-phase protein array.
abstract: The comprehensive and quantitative analysis of the protein phosphorylation patterns in different cellular context is of considerable and general interest. The ability to quantify phosphorylation of discrete signalling proteins in large collections of biological samples would greatly favour the development of systems biology in the field of cell signalling. Reverse-phase protein array (RPPA) potentially represents a very attractive approach to map signal transduction networks with high throughput. In the present report, we describe an improved detection method for RPPA combining near-infrared with one or two rounds of tyramide-based signal amplification. The LOQ was lowered from 6.84 attomoles with a direct detection protocol to 0.21 attomole with two amplification steps. We validated this method in the context of intracellular signal transduction triggered by follicle-stimulating hormone in HEK293 cells. We consistently detected phosphorylated proteins in the sub-attomole range from less than 1 ng of total cell extracts. Importantly, the method correlated with Western blot analysis of the same samples while displaying excellent intra- and inter-slide reproducibility. We conclude that RPPA combined with amplified near-infrared detection can be used to capture the subtle regulations intrinsic to signalling network dynamics at an unprecedented throughput, from minute amounts of biological samples.
authors.affiliation: BIOS group, INRA, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Dupuy
authors.forename: Christophe
authors.initials: C
authors.lastname: Gauthier
authors.forename: Guillaume
authors.initials: G
authors.lastname: Durand
authors.forename: Astrid
authors.initials: A
authors.lastname: Musnier
authors.forename: Domitille
authors.initials: D
authors.lastname: Heitzler
authors.forename: Adrien
authors.initials: A
authors.lastname: Herledan
authors.forename: Vehary
authors.initials: V
authors.lastname: Sakanyan
authors.forename: Pascale
authors.initials: P
authors.lastname: Crépieux
authors.forename: Eric
authors.initials: E
authors.lastname: Reiter
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D005453:Fluorescence; D005640:Follicle Stimulating Hormone / Q000032:analysis / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D019265:Spectroscopy, Near-Infrared
chemical_list: D011506:Proteins; D005640:Follicle Stimulating Hormone