title: Integrated platform with a combination of online digestion and (18)O labeling for proteome quantification via an immobilized trypsin microreactor.
abstract: A novel automated integrated platform for quantitative proteome analysis was established with a combination of online digestion of proteins and in situ(18)O labeling by an immobilized enzyme reactor (IMER); digests were captured and desalted by a C18 trap column, and peptides were analyzed by nanoRPLC-ESI-MS/MS. Bovine serum albumin (BSA) was used to evaluate the performance of the developed platform. Compared with traditional offline methods, not only the digestion and labeling time was shortened from 36 h to just 1 h, but also the labeling efficiency was improved from 95% to 99%. Furthermore, the back-exchange from (18)O to (16)O could also be efficiently avoided by the use of IMER. The platform was further evaluated by the quantitative analysis of 100 ng (18)O and (16)O online labeled yeast sample with a mixing ratio of 1 : 1, and the results showed significantly improved sensitivity and reproducibility, as well as improved quantitative accuracy than offline method. With these advantages, the integrated platform was finally applied to the quantitative profiling of 100 ng proteins extracted from two mouse hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastases rates, and ten differentially expressed proteins were successfully found, most of which were related to tumorigenesis and tumor metastasis. All these results demonstrate that the developed integrated platform can provide a new way for high efficiency (18)O labeling and the quantitative analysis of trace amounts of sample with high accuracy and high reproducibility.
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, 116023, China. lihuazhang@dicp.ac.cn.
authors.forename: Shen
authors.initials: S
authors.lastname: Zhang
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Baofeng
authors.initials: B
authors.lastname: Zhao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D008113:Liver Neoplasms / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation; D053719:Tandem Mass Spectrometry / Q000295:instrumentation; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D020543:Proteome; D012710:Serum Albumin, Bovine; D014357:Trypsin