title: A fast workflow for identification and quantification of proteomes.
abstract: The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Ding
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.forename: Junying
authors.initials: J
authors.lastname: Wei
authors.forename: Wanlin
authors.initials: W
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Mingwei
authors.initials: M
authors.lastname: Liu
authors.forename: Tianyi
authors.initials: T
authors.lastname: Fu
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Lu
authors.forename: Lei
authors.initials: L
authors.lastname: Song
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Cheng
authors.initials: C
authors.lastname: Chang
authors.forename: Yangjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Ma
authors.forename: Lai
authors.initials: L
authors.lastname: Wei
authors.forename: Anna
authors.initials: A
authors.lastname: Malovannaya
authors.forename: Lijun
authors.initials: L
authors.lastname: Jia
authors.forename: Bei
authors.initials: B
authors.lastname: Zhen
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000494:pharmacology; D006367:HeLa Cells; D056945:Hep G2 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011743:Pyrimidines / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
chemical_list: D003517:Cyclopentanes; D020543:Proteome; D011743:Pyrimidines; C539933:pevonedistat