title: Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome.
abstract: The development of high-resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse-phase long column (100 μm × 150 cm, 5 μm C18 beads) coupled to Q Exactive MS. The column was capable of achieving a peak capacity of ∼700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was ∼6 μg of peptides, although the column allowed loading as many as 20 μg. Gas-phase fractionation of peptide ions further increased the number of peptide identification by ∼10%. Moreover, the combination of basic pH LC prefractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a post-mortem brain sample of Alzheimer's disease. Because deep RNA sequencing of the same specimen suggested that ∼16,000 genes were expressed, the current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.
authors.affiliation: Department of Structural Biology, ‡St. Jude Proteomics Facility, and §̂Department of Developmental Neurobiology, St. Jude Children's Research Hospital , 262 Danny Thomas Place, Memphis, Tennessee 38105, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Yanling
authors.initials: Y
authors.lastname: Yang
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Bai
authors.forename: Xusheng
authors.initials: X
authors.lastname: Wang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Tan
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Beach
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry / Q000502:physiology*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020543:Proteome
keywords: AD proteome; long LC column; mass spectrometry