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      Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.

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          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.

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          Author and article information

          Journal
          Proteomics
          Proteomics
          Wiley
          1615-9861
          1615-9853
          May 2011
          : 11
          : 10
          Affiliations
          [1 ] Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
          Article
          NIHMS299666
          10.1002/pmic.201000722
          3120047
          21500348
          7c151958-7b18-4af4-a17b-d1d566329bb0
          Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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