title: Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.
abstract: In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Yuexi
authors.initials: Y
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Marina A
authors.initials: MA
authors.lastname: Gritsenko
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Wang
authors.forename: Therese
authors.initials: T
authors.lastname: Clauss
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Shen
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.forename: Daniel
authors.initials: D
authors.lastname: Lopez-Ferrer
authors.forename: Theresa
authors.initials: T
authors.lastname: Reno
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Richard L
authors.initials: RL
authors.lastname: Klemke
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D001940:Breast / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D016000:Cluster Analysis; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D005561:Formates / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D005561:Formates; D010446:Peptide Fragments; D020543:Proteome; C030544:formic acid; D014508:Urea; D014357:Trypsin; C032159:acetonitrile