title: Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins.
abstract: We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC-MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.
authors.affiliation: Yale/NIDA Neuroproteomics Center, New Haven, CT, USA; W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT, USA; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Colangelo
authors.forename: Gordana
authors.initials: G
authors.lastname: Ivosev
authors.forename: Lisa
authors.initials: L
authors.lastname: Chung
authors.forename: Thomas
authors.initials: T
authors.lastname: Abbott
authors.forename: Mark
authors.initials: M
authors.lastname: Shifman
authors.forename: Fumika
authors.initials: F
authors.lastname: Sakaue
authors.forename: David
authors.initials: D
authors.lastname: Cox
authors.forename: Robert R
authors.initials: RR
authors.lastname: Kitchen
authors.forename: Lyle
authors.initials: L
authors.lastname: Burton
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Tate
authors.forename: Erol
authors.initials: E
authors.lastname: Gulcicek
authors.forename: Ron
authors.initials: R
authors.lastname: Bonner
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.forename: Angus C
authors.initials: AC
authors.lastname: Nairn
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Williams
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification; D057907:Post-Synaptic Density / Q000737:chemistry; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics; D051381:Rats; D013569:Synapses / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D009419:Nerve Tissue Proteins; D020543:Proteome
keywords: Multiple reaction monitoring; Postsynaptic density; Proteomics database; Targeted proteomics; Technology