title: In situ sample processing approach (iSPA) for comprehensive quantitative phosphoproteome analysis.
abstract: Current sample preparation protocols for quantitative phosphoproteome analysis are tedious and time-consuming. Here, a facile in situ sample processing approach (iSPA) is developed by using macroporous Ti(IV)-IMAC microspheres as the preparation "beds", where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment, and isotope labeling of phosphopeptides are performed in situ sequentially. As a result of the in situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided, and the sample loss and contamination could be greatly reduced. Thus, better sensitivity and accuracy for the quantitative phosphoproteome analysis were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). In total, 8548 phosphorylation sites were confidently quantified from three replicate analyses of 0.5 mg of human liver protein extracts.
authors: CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors: Junfeng
authors: J
authors: Huang
authors: Hongqiang
authors: H
authors: Qin
authors: Jing
authors: J
authors: Dong
authors: Chunxia
authors: C
authors: Song
authors: Yangyang
authors: Y
authors: Bian
authors: Mingming
authors: M
authors: Dong
authors: Kai
authors: K
authors: Cheng
authors: Fangjun
authors: F
authors: Wang
authors: Deguang
authors: D
authors: Sun
authors: Liming
authors: L
authors: Wang
authors: Mingliang
authors: M
authors: Ye
authors: Hanfa
authors: H
authors: Zou
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008113:Liver Neoplasms / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D010750:Phosphoproteins; D020543:Proteome