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Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2019, 91, 17, 11085-11091
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Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics

  • Yehia Z. Baghdady
    Yehia Z. Baghdady
    Department of Chemistry & Biochemistry, The University of Texas Arlington, Arlington, Texas 76019-0065, United States
    More by Yehia Z. Baghdady
  •  and 
  • Kevin A. Schug*
    Kevin A. Schug
    Department of Chemistry & Biochemistry, The University of Texas Arlington, Arlington, Texas 76019-0065, United States
    *Phone: 817-272-3541. E-mail: [email protected]. Corresponding author address: 700 Planetarium Pl., Box 19065, Arlington, TX 76019.
    More by Kevin A. Schug
    Orcidhttp://orcid.org/0000-0003-4817-9508
Cite this: Anal. Chem. 2019, 91, 17, 11085–11091
Publication Date (Web):August 1, 2019
https://doi.org/10.1021/acs.analchem.9b01665
Copyright © 2019 American Chemical Society
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    Abstract

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    A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on–off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

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