title: Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysis.
abstract: Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses.
authors.affiliation: Department of Life Sciences Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Intoh
authors.forename: Akira
authors.initials: A
authors.lastname: Kurisaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fukuda
authors.forename: Makoto
authors.initials: M
authors.lastname: Asashima
mesh_terms: D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D053595:Embryonic Stem Cells / Q000737:chemistry / Q000166:cytology; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D051379:Mice; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D012709:Serum Albumin; D014357:Trypsin